bluuebunny/crossref_metadata_2025 huggingface dataset:
gamma-Aminobutyric acid (GABA)-induced currents of skate Muller (glial) cells are mediated by neuronal-like GABAA receptors.
Authors: Malchow R P , Qian H H , Ripps H
Published: May 2006

Radial glia (Muller cells) of the vertebrate retina appear to be intimately involved in regulating the actions of amino acid neurotransmitters. One of the amino acids thought to be important in mediating retinal information flow is gamma-aminobutyric acid (GABA). The findings of this study indicate that enzymatically isolated skate Muller cells are depolarized by GABA and the GABAA agonist muscimol and that the actions of these agents are reduced by bicuculline and picrotoxin. Membrane currents induced by GABA under voltage clamp were dose dependent, were associated with an increase in membrane conductance, and showed marked desensitization when the concentration of GABA exceeded 2.5 microM. The responses had a reversal potential close to that calculated for chloride, indicating that the currents were generated by ions passing through channels. These data support the view that skate Muller cells possess functional GABAA receptors. The presence of such receptors on retinal glia may have important implications for the role of Muller cells in maintaining the constancy of the extracellular milieu, for neuron-glia interactions within the retina, and for theories concerning the generation of the electroretinogram.
Correlation of paramagnetic states and molecular structure in bacterial photosynthetic reaction centers: the symmetry of the primary electron donor in Rhodopseudomonas viridis and Rhodobacter sphaeroides R-26.
Authors: Norris J R , Budil D E , Gast P , Chang C H , el-Kabbani O , Schiffer M
Published: May 2006

The orientation of the principal axes of the primary electron donor triplet state measured in single crystals of photosynthetic reaction centers is compared to the x-ray structures of the bacteria Rhodobacter (Rb.) sphaeroides R-26 and Rhodopseudomonas (Rps.) viridis. The primary donor of Rps. viridis is significantly different from that of Rb. sphaeroides. The measured directions of the axes indicate that triplet excitation is almost completely localized on the L-subunit half of the dimer in Rps. viridis but is more symmetrically distributed (approximately 63% on the L half of the special pair and approximately 37% on the M half) on the dimeric donor in Rb. sphaeroides R-26. The large reduction of the zero field splitting parameters relative to monomeric bacteriochlorophyll triplet in vitro suggests significant participation of asymmetrical charge transfer electronic configurations in the special pair triplet state of both organisms (approximately 23% in Rps. viridis and approximately 13% in Rb. sphaeroides).
Phosphorylation-dependent activation of the adenovirus-inducible E2F transcription factor in a cell-free system.
Authors: Bagchi S , Raychaudhuri P , Nevins J R
Published: May 2006

Adenovirus infection induces a large increase in the DNA binding activity of a cellular transcription factor that is utilized by the viral E2 promoter and termed E2F. Using cell-free extracts, we have developed an assay for the in vitro activation of DNA binding activity of E2F. E2F activity is undetectable in HeLa extracts but upon incubation with a fraction from adenovirus-infected cells, there is an ATP-dependent increase in E2F DNA binding activity. This increase does not occur using an equivalent fraction from dl312 (E1A-)-infected cells. Incubation of E2F with phosphatase inactivates E2F binding activity. Incubation of the phosphatase-inactivated E2F with an infected cell fraction restores E2F activity as does incubation with a known protein kinase. In contrast, incubation with an extract from mock-infected cells does not restore activity. We conclude that the DNA binding activity of E2F is regulated by phosphorylation in an E1A-dependent manner.
The PapG adhesin of uropathogenic Escherichia coli contains separate regions for receptor binding and for the incorporation into the pilus.
Authors: Hultgren S J , Lindberg F , Magnusson G , Kihlberg J , Tennent J M , Normark S
Published: May 2006

Most uropathogenic strains of Escherichia coli produce heteropolymeric organelles, known as P pili, that bind to the globoseries of glycolipids present in the urinary tract. The formation of a P pilus is the result of a family of related proteins being coordinately assembled into the structure in a defined order with the adhesin located exclusively at the tip. The preassembled digalactoside alpha-D-galactopyranosyl-(1----4)-beta-D-galactopyranose-binding adhesin was purified to homogeneity from the periplasmic space in a complex with the periplasmic assembly protein PapD by affinity chromatography to alpha-D-galactopyranosyl-(1----4)-beta-D-galactopyranose-Sepharose. A receptor-binding domain was mapped to the amino-terminal half of the adhesin. The interaction of PapD with PapG, which was required for the incorporation of the adhesin into the pilus, was found to protect PapG from proteolytic cleavages and enhanced the processing of the PapG signal peptide. A preassembly domain necessary for forming a complex with PapD was mapped to the carboxyl terminus of PapG.
Two peptides related to platelet-derived growth factor are present in human wound fluid.
Authors: Matsuoka J , Grotendorst G R
Published: May 2006

Platelet-derived growth factor (PDGF) has been suggested to play an important role in wound healing. In this study wound fluid collected from patients after radical mastectomy was analyzed for PDGF-related factors. Analysis revealed the presence of two PDGF-related peptides of 16-17 kDa and 34-36 kDa. These PDGF-related peptides were purified by using anti-PDGF immunoaffinity techniques and found to possess both chemotactic and mitogenic activities for NIH 3T3 cells in vitro. Surprisingly, we could not detect authentic native PDGF (30 kDa) or PDGF A or B chain in any of the wound-fluid samples examined. These results indicate that PDGF-related peptides are present in normal human wound fluid, whereas PDGF A- and B-chain peptides are absent or only present in trace amounts relative to the total PDGF-related biological activity.
Oxygen permeability of phosphatidylcholine--cholesterol membranes.
Authors: Subczynski W K , Hyde J S , Kusumi A
Published: May 2006

Oxygen transport in phosphatidylcholine-cholesterol membranes has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed at various distances from the membrane surface using long-pulse saturation recovery ESR techniques. The collision rate was estimated for tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameter across the membrane were obtained as a function of cholesterol mol fraction and temperature in L-alpha-dimyristoylphosphatidylcholine ([ Myr2]PtdCho) and L-alpha-dioleoylphosphatidylcholine ([ Ole2]PtdCho) membranes. Membrane oxygen permeability coefficients were estimated from oxygen transport parameter profiles. At approximately 30 degrees C, the oxygen permeability coefficients in the presence and absence of 50 mol % cholesterol are 22.7 and 125.2 cm/s, respectively, for [Myr2]PtdCho membranes, and 54.7 and 114.2 cm/s, respectively, for [Ole2]PtdCho membranes (compared with 60-80 cm/s for water layers with the same thicknesses as the membranes). The major results in the liquid-crystalline phase are as follows: (i) In the absence of cholesterol, membranes are not barriers to oxygen transport. (ii) Addition of 50 mol % cholesterol decreases oxygen permeability by a factor of approximately 5 and approximately 2.5 in [Myr2]PtdCho and [Ole2]PtdCho membranes, respectively. The resistance to oxygen transport is located in and near the polar headgroup regions in the membrane. (iii) Cholesterol increases oxygen transport in the central regions of [Ole2]PtdCho membranes.
Estimation of glucose carbon recycling in children with glycogen storage disease: A 13C NMR study using [U-13C]glucose.
Authors: Kalderon B , Korman S H , Gutman A , Lapidot A
Published: May 2006

A stable isotope procedure to estimate hepatic glucose carbon recycling and thereby elucidate the mechanism by which glucose is produced in patients lacking glucose 6-phosphatase is described. A total of 10 studies was performed in children with glycogen storage disease type I (GSD-I) and type III (GSD-III) and control subjects. A primed dose-constant nasogastric infusion of D-[U-13C]glucose (greater than 99% 13C-enriched) or an infusion diluted with nonlabeled glucose solution was administered following different periods of fasting. Hepatic glucose carbon recycling was estimated from 13C NMR spectra. The recycling parameters were derived from plasma beta-glucose C-1 splitting pattern, doublet/singlet values of plasma glucose C-1 in comparison to doublet/singlet values of known mixtures of [U-13C]glucose and unlabeled glucose as a function of 13C enrichment of glucose C-1. The fractional glucose C-1 enrichment of plasma glucose samples was analyzed by 1H NMR spectroscopy and confirmed by gas chromatography/mass spectroscopy. The values obtained for GSD-I patients coincided with the standard [U-13C]glucose dilution curve. These results indicate that the plasma glucose of GSD-I subjects comprises only a mixture of 99% 13C-enriched D-[U-13C]glucose and unlabeled glucose but lacks any recycled glucose. Significantly different glucose carbon recycling values were obtained for two GSD-III patients in comparison to GSD-I patients. Our results eliminate a mechanism for glucose production in GSD-I children involving gluconeogenesis. However, glucose release by amylo-1,6-glucosidase activity would result in endogenous glucose production of non-13C-labeled and nonrecycled glucose carbon, as was found in this study. In GSD-III patients gluconeogenesis is suggested as the major route for endogenous glucose synthesis. The contribution of the triose-phosphate pathway in these patients has been determined. The significant difference of the glucose C-1 splitting pattern in plasma GSD-III and control subjects, in comparison to GSD-I plasma, can be used as a parameter for estimating glucose recycling. This approach can be developed as a noninvasive diagnostic test for inborn enzymatic defects involving gluconeogenesis.
Identification of a high-virulence clone of type III Streptococcus agalactiae (group B Streptococcus) causing invasive neonatal disease.
Authors: Musser J M , Mattingly S J , Quentin R , Goudeau A , Selander R K
Published: May 2006

Chromosomal genotypes of 128 isolates of six serotypes (Ia, Ib, Ic, II, Ic/II, and III) of Streptococcus agalactiae (group B Streptococcus) recovered predominantly from human infants in the United States were characterized by an analysis of electrophoretically demonstrable allelic profiles at 11 metabolic enzyme loci. Nineteen distinctive electrophoretic types (ETs), representing multilocus clonal genotypes, were identified. Mean genetic diversity per locus among ETs of isolates of the same serotype was, on average, nearly equal to that in all 19 ETs. Cluster analysis of the ETs revealed two primary phylogenetic divisions at a genetic distance of 0.65. A single clone (ET 1) represented by 40 isolates expressing type III antigen formed division I. Division II was composed of 18 ETs in three major lineages diverging from one another at distances greater than 0.35 and included strains of all six antigenic classes. The type III organisms in division I produce more extracellular neuraminidase and apparently are more virulent than the type III strains in division II, which are related to strains of other serotypes that cause disease much less frequently. The existence of this unusually virulent clone accounts, in major part, for the high morbidity and mortality associated with infection by type III organisms.
A cell line producing recombinant nerve growth factor evokes growth responses in intrinsic and grafted central cholinergic neurons.
Authors: Ernfors P , Ebendal T , Olson L , Mouton P , Strömberg I , Persson H
Published: May 2006

The rat beta nerve growth factor (NGF) gene was inserted into a mammalian expression vector and cotransfected with a plasmid conferring resistance to neomycin into mouse 3T3 fibroblasts. From this transfection a stable cell line was selected that contains several hundred copies of the rat NGF gene and produces excess levels of recombinant NGF. Such genetically modified cells were implanted into the rat brain as a probe for in vivo effects of NGF on central nervous system neurons. In a model of the cortical cholinergic deficits in Alzheimer disease, we demonstrate a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain cholinergic neurons, as well as stimulation of fiber formation by intact adult intrinsic cholinergic circuits in the cerebral cortex. Adult cholinergic interneurons in intact striatum also sprout vigorously toward implanted fibroblasts. Our results suggest that this model has implications for future treatment of neurodegenerative diseases.
Derivation of an infectious viral RNA by autolytic cleavage of in vitro transcribed viral cDNAs.
Authors: Dzianott A M , Bujarski J J
Published: May 2006

An in vitro transcription system has been developed that utilizes self-processing to adjust 3' termini in transcribed viral RNAs. Transcription vectors were constructed by ligating a cDNA "cassette" containing self-cleavage sequences from the satellite RNA of tobacco ringspot virus (STobRV RNA) with the 3' end of brome mosaic virus (BMV) cDNA clones. Transcripts were obtained either from templates linearized at positions located downstream from the cassette or from nonlinearized plasmids. In both cases, a spontaneous self-cleavage reaction produced BMV RNAs that contained 19 heterologous nucleotides at their 3' ends. These RNAs were biologically active, indicating that 19 extra nucleotides did not remove the infectivity. The progeny BMV RNA isolated from infected plants did not retain the 19-base heterologous sequence. BMV RNAs containing longer 3' heterologous sequences were not infectious. The STobRV self-cleavage cassette is potentially applicable to a variety of RNA systems.
Structural characterization of the rat malic enzyme gene.
Authors: Morioka H , Magnuson M A , Mitsuhashi T , Song M K , Rall J E , Nikodem V M
Published: May 2006

We have identified and characterized lambda bacteriophage clones containing genomic DNA encoding rat malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating); EC 1.1.1.40]. The malic enzyme gene is unexpectedly large, spanning at least 95 kilobases. It is divided into 14 exons that range in size from 76 to 1513 base pairs. The sizes and boundaries of the exons were determined by Southern blotting and DNA sequencing. The sequences at the 5' and 3' ends of each intron conformed to the consensus sequence for mammalian introns. S1 nuclease and primer-extension assays showed that transcription of the malic enzyme gene initiates at multiple sites, the strongest one at position -31 relative to the ATG. "TATA and CCAAT box" homologies are not present in the proximal promoter region. Analysis of the 3' end of the gene showed that the utilization of alternate polyadenylylation signals in exon 14 results in two mRNAs with 3' untranslated regions of 345 and 1345 nucleotides, respectively.
Membranous intermediates in endocytosis are labile, as shown in a temperature-sensitive mutant.
Authors: Kessell I , Holst B D , Roth T F
Published: May 2006

Membranous tubules, especially prevalent in mammalian absorptive epithelia and insect oocytes, are one of the pleomorphic endocytic compartments that have a role in receptor-mediated endocytosis. To determine whether these tubules are evanescent, and to investigate their temporal relationships with other endocytic intermediates, we studied these tubules during oocyte vitellogenesis in the temperature-sensitive mutant Drosophila melanogaster, shibiretsl. Raising the temperature of shibire oocytes for 1 min from 19 degrees C to 29 degrees C caused a loss of these membranous tubules. The percentage of membrane in tubules decreased from 36% at 19 degrees C to 1.5% after 5 min at 29 degrees C. Concomitantly, the amount of surface membrane increased from 64% at 19 degrees C to 98% after 5 min at 29 degrees C, causing surface membrane invaginations to extend deeper into the cortex. At 29 degrees C the cytoplasmic face of the plasma membrane was studded with coated pits, and the extracellular face was coated with electron-dense material. Return from 29 degrees C to either 19 degrees C or 26 degrees C for 1-2 min produced a rapid reappearance of tubules containing extracellular horseradish peroxidase in the cortex. These data suggest that tubular intermediates are evanescent structures, and that temperature shock (i) rapidly blocks their formation from the plasma membrane, (ii) causes existing tubules to rapidly recycle to the plasma membrane, and (iii) is rapidly reversed, as newly formed tubules derive their membrane and content from the cell surface.
Mitosis-specific monoclonal antibody MPM-2 inhibits Xenopus oocyte maturation and depletes maturation-promoting activity.
Authors: Kuang J , Zhao J , Wright D A , Saunders G F , Rao P N
Published: May 2006

MPM-2, a monoclonal antibody specific for cells in mitosis, recognizes a family of proteins that share a common phosphorylated epitope. In this study we have shown that during the maturation of Xenopus laevis oocytes induced by progesterone, phosphorylation of MPM-2 antigens coincided with the appearance of MPF activity. When MPM-2 (0.7-1.4 micrograms per oocyte) was injected into oocytes prior to progesterone stimulation, MPF activity failed to appear and induction of maturation was inhibited as judged by both germinal-vesicle breakdown and white-spot formation. Further, MPM-2 was able to neutralize as well as immunodeplete MPF activity from mitotic HeLa cell and mature oocyte extracts. These results suggest that MPM-2 recognizes either MPF itself or a protein(s) that regulates MPF activity and that the kinase that phosphorylates MPM-2 antigens may be a key component in the regulation of M-phase induction.
Polymorphism for the number of tandemly multiplicated glycerol-3-phosphate dehydrogenase genes in Drosophila melanogaster.
Authors: Takano T , Kusakabe S , Koga A , Mukai T
Published: May 2006

A 26-kilobase-pair region encompassing the sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) locus in Drosophila melanogaster from two natural populations in Japan was surveyed by restriction mapping. Both tandem duplications and triplications in this region were found in both populations. Detailed analysis of 86 chromosome 2 lines revealed restriction site and allozyme polymorphisms in the transcriptional unit: two restriction sites and the allozymes [fast (F) or slow (S)] were polymorphic among both duplication-bearing chromosomes and those carrying the standard sequence. This finding suggests recurrent recombination and/or gene conversion in this 5-kilobase-pair region. The differences observed for restriction site and allozyme haplotypes among the triplicated sequence both within and between populations, together with the distribution in natural populations, suggest a relatively recent ancestry of the triplication events and an independent origin in respective populations. Such events may represent the process of the formation of multigene families [compare Ohta, T. (1987) Genetics 115, 207-213]. Finally, the evolution of this type of polymorphism is discussed.
Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3' end of the acceptor arm: a model of the tRNA-ribosome complex.
Authors: Wower J , Hixson S S , Zimmermann R A
Published: May 2006

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine (2N3A) at position 73 or 76 have been crosslinked to the peptidyl site of Escherichia coli ribosomes. Covalent tRNA-ribosome attachment was dependent upon the replacement of adenosine by 2N3A in the tRNA, irradiation with 300-nm light, and the presence of poly(U). In all cases, the modified tRNAs became crosslinked exclusively to 50S ribosomal subunits. While the tRNA derivative containing 2N3A at position 73 labeled only protein L27, that containing 2N3A at position 76 labeled proteins L15, L16, and L27 as well as a segment of the 23S rRNA. The site of crosslinking in the rRNA was identified as guanosine-1945, which lies within a highly conserved sequence adjacent to a number of modified bases and has not until now been identified at the peptidyltransferase center. On the basis of these results, and previously reported crosslinks from tRNA containing 8-azidoadenosine in the 3'-terminal -A-C-C-A sequence [Wower, J., Hixson, S. S. & Zimmermann, R. A. (1988) Biochemistry 27, 8114-8121], we propose a model for the arrangement of tRNA molecules at the peptidyl and aminoacyl sites that is consistent with most of the information available about the location of the peptidyltransferase center and the decoding domain of the E. coli ribosome.
Altered membrane association of p60v-src and a murine 63-kDa N-myristoyl protein after incorporation of an oxygen-substituted analog of myristic acid.
Authors: Heuckeroth R O , Gordon J I
Published: May 2006

A number of viral and cellular proteins contain covalently bound lipid. In a subset of these acyl proteins, the 14-carbon saturated fatty acid myristic acid is attached through an amide linkage to an NH2-terminal glycine residue. Myristoyl-CoA:protein N-myristoyltransferase (NMT) transfers the myristoyl moiety from myristoyl-CoA to these nascent proteins and is highly selective for fatty acid chain length. We have found that 10-(propoxy)decanoyl-CoA (11-oxymyristoyl-CoA), an analog of myristic acid with reduced hydrophobicity, acts as a substrate for NMT both in vitro and in vivo. Comparison of the in vitro kinetic properties of a number of synthetic octapeptide substrates of NMT using myristoyl-CoA or 11-oxymyristoyl-CoA indicated that there is an interaction between the acyl-CoA and peptide binding sites of this acyltransferase. Peptide catalytic efficiency with 11-oxymyristoyl-CoA was reduced relative to that with myristoyl-CoA, but the extent of the reduction varied widely among the octapeptides tested. These in vitro data accurately predicted that only a subset of myristoyl proteins synthesized in Saccharomyces cerevisiae and a murine myocyte-like cell line (BC3H1) would incorporate 11-oxy[3H]myristate. Substitution of the myristoyl moiety by the 11-oxymyristoyl moiety does not significantly affect the membrane association of most N-myristoyl proteins. However, for the tyrosine kinase p60v-src and a 63-kDa N-myristoyl protein in BC3H1 cells, analog incorporation results in marked redistribution from the membrane to the cytosolic fraction. These studies demonstrate the utility of heteroatom-containing analogs for analysis of the role of myristate in acyl protein targeting. The sequence-specific nature of analog incorporation and the protein-specific effects on membrane association suggests that these compounds may represent a useful class of antiviral and antitumor agents.
Unusually stable helix formation in short alanine-based peptides.
Authors: Marqusee S , Robbins V H , Baldwin R L
Published: May 2006

Short, 16-residue, alanine-based peptides show stable alpha-helix formation in H2O. This result is surprising when contrasted with the classical view that regards the alpha-helix as a marginally stable structure in H2O and considers short helices unstable. The alanine-based peptides are solubilized by insertion of three or more residues of a single charge type, lysine (+) or glutamic acid (-). The results cannot be explained by helix stabilization resulting from concentration-dependent association or by the interaction of charged residues with the helix dipole. Our results are not predicted by the parameters for alanine and lysine that have been determined by the "host-guest" method: these parameters predict that a 16-residue peptide should not show measurable alpha-helix formation. Analysis of the role of the hydrophobic interaction in alpha-helix formation [Richards, F.M. & Richmond, T. (1978) in Molecular Interactions and Activity in Proteins, Ciba Foundation Symposium 60, ed. Wolstenholme, G.E. (Excepta Medica Amsterdam), pp. 23-25] does not show an unusually strong hydrophobic interaction in a helical block of alanine residues. The likely explanation for our results is, therefore, that individual alanine residues have a high helical potential. It is not yet known whether any other amino acids show this property, and the origin of this property is also unknown.
Multiplex gene regulation: a two-tiered approach to transgene regulation in transgenic mice.
Authors: Byrne G W , Ruddle F H
Published: May 2006

Transgenic mice have been used to study gene function and regulation by introducing inducible or tissue-specific transgenes. This approach is generally limited to studying gene function in adult mice since ectopic expression of many interesting genes is disease causing or may be lethal to the developing embryo. To extend the utility of the transgenic mouse system to the early stages of embryogenesis, we have developed a two-tiered method of gene regulation to control transgene expression. Our multiplex gene regulatory system (MGR) allows the establishment of transgenic lines that harbor inducible potentially lethal transgenes. These inducible transgenes are activated only when mated to a second transgenic animal. Induction in the MGR system provides a high degree of temporal and spatial control over transgene expression and should be suitable for engineering "gain of function mutations" for many developmental genes.
The homeotic gene spalt (sal) evolved during Drosophila speciation.
Authors: Reuter D , Schuh R , Jäckle H
Published: May 2006

The region-specific homeotic gene spalt (sal) acts in two separate domains in the head and tail region of the Drosophila melanogaster embryo. Based on comparative morphology, sal is likely to be involved in the establishment of the head during the evolution of invertebrates and thus, it should be conserved. We have analyzed the conservation of the segmentation genes Krüppel (Kr) and even-skipped (eve) in parallel with sal coding sequences in several Drosophila species that are evolutionarily separated by up to 60 million years. To our surprise, sal sequences appear to be conserved in the Sophophora subgenus of the Drosophila genus but not in the Drosophila subgenus. On the other hand, the segmentation and other homeotic genes are conserved in the Drosophila subgroup as well. Our data suggest that sal encodes an accessory function that evolved relatively late during Drosophila speciation rather than playing a fundamental evolutionary role similar to that of other homeotic genes.
UV induction of coliphage 186: prophage induction as an SOS function.
Authors: Lamont I , Brumby A M , Egan J B
Published: May 2006

Our results show that UV induction of the 186 prophage depends upon the phage function Tum, with the mutant phenotype of turbid plaques on mitomycin plates and the expression of which is controlled by the host LexA protein. Tum function, encoded near the right-hand end of the coliphage 186 chromosome, is under the control of promoter p95. This promoter is overlapped by a sequence closely related to the consensus sequence of the LexA-binding site. It is proposed that inactivation of LexA after UV irradiation (or by genetic means) leads to prophage induction by permitting expression of Tum which, by unknown means, induces prophage. This mechanism is basically different from that seen with the UV-inducible lambdoid coliphages, which are not regulated by LexA.
Structure of the human retinoblastoma gene.
Authors: Hong F D , Huang H J , To H , Young L J , Oro A , Bookstein R , Lee E Y , Lee W H
Published: May 2006

Complete inactivation of the human retinoblastoma gene (RB) is believed to be an essential step in tumorigenesis of several different cancers. To provide a framework for understanding inactivation mechanisms, the structure of RB was delineated. The RB transcript is encoded in 27 exons dispersed over about 200 kilobases (kb) of genomic DNA. The length of individual exons ranges from 31 to 1889 base pairs (bp). The largest intron spans greater than 60 kb and the smallest one has only 80 bp. Deletion of exons 13-17 is frequently observed in various types of tumors, including retinoblastoma, breast cancer, and osteosarcoma, and the presence of a potential "hot spot" for recombination in the region is predicted. A putative "leucine-zipper" motif is exclusively encoded by exon 20. The detailed RB structure presented here should prove useful in defining potential functional domains of its encoded protein. Transcription of RB is initiated at multiple positions and the sequences surrounding the initiation sites have a high G + C content. A typical upstream TATA box is not present. Localization of the RB promoter region was accomplished by utilizing a heterologous expression system containing a bacterial chloramphenicol acetyltransferase gene. Deletion analysis revealed that a region as small as 70 bp is sufficient for RB promoter activity, similar to other previously characterized G + C-rich gene promoters. Several direct repeats and possible stem-and-loop structures are found in the promoter region. No enhancer element was detected within the 7.3 kb of upstream sequence studied. Several features of the RB promoter are reminiscent of the characteristics associated with many "housekeeping" genes, consistent with its ubiquitous expression pattern.
Loss of HLA-A,B,C allele products and lymphocyte function-associated antigen 3 in colorectal neoplasia.
Authors: Smith M E , Marsh S G , Bodmer J G , Gelsthorpe K , Bodmer W F
Published: May 2006

The expression of HLA-A,B,C antigens and lymphocyte function-associated antigen 3 in human colorectal adenomas and adenocarcinomas was studied by immunohistochemistry. None of 10 adenomas and only 1 of 30 carcinomas had lost expression of all HLA-A,B,C molecules. On the other hand, focal loss of an HLA-B product was seen in 2 of the adenomas, and complete losses of tumor cell HLA-A2 (in 7 of 13 cases), HLA-Bw4 (in 4 of 13 cases), and HLA-A3 (in 1 of 6 cases) were seen in the carcinomas. No complete losses of HLA-A1 (in 6 cases) or HLA-Bw6 (in 22 cases) occurred in the carcinomas. In addition, 1 of 20 adenocarcinomas totally lacked lymphocyte function-associated antigen 3. Because a loss of tumor cell HLA-A,B,C antigen or lymphocyte function-associated antigen 3 could be selected for through an advantage in escape from cytotoxic T-lymphocyte attack, our results suggest that immunoselection may be a more important mechanism in tumor progression than has previously been assumed.
Retinoid receptor antisense DNAs inhibit alkaline phosphatase induction and clonogenicity in malignant keratinocytes.
Authors: Cope F O , Wille J J
Published: May 2006

Antisense oligodeoxynucleotides [oligo(dN)s] corresponding to human cellular retinol-binding protein I (cRBP) and human nuclear retinoic acid receptor alpha (hnRAR) were synthesized. Exposure of human malignant keratinocytes to these oligo(dN)s significantly attenuated the level of cytoplasmic cRBP and hnRAR in a concentration- and time-dependent manner. Further, the induction of alkaline phosphatase by retinol in these cells was blocked by treatment with 30 microM antisense oligo(dN) to cRBP or hnRAR but not by 30 microM of sense oligo(dN) to cRBP. Antisense oligo(dN) treatments concomitantly induced cell rounding, loss of cell-cell attachment, and cell adhesion to the substratum. By contrast, treatment of cells with an anticytokinetic agent, cytochalasin B, or with a cytostatic concentration of sodium azide failed to reduce cytoplasmic cRBP or hnRAR from nuclear extracts, even though antisense oligo(dN)-like changes in cell morphology were observed. Treatment of the cells for greater than 2.75 hr with 20-40 microM of either antisense oligo(dN) also led to the loss of clonogenic potential. These results show that both cytoplasmic and nuclear receptors for retinoids are important in the transduction of a retinoid signal response critical to cellular growth and differentiation. Our findings also suggest that defined genes, which are specified by retinoids and their receptors, may account for the pleiotropic effect of vitamin A compounds.
Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiated in situ with ultraviolet light.
Authors: Freeman S E , Hacham H , Gange R W , Maytum D J , Sutherland J C , Sutherland B M
Published: May 2006

The UV components of sunlight are believed to be a major cause of human skin cancer, and DNA is thought to be the principal molecular target. Alterations of the intensity and wavelength distribution of solar UV radiation reaching the surface of the earth, for example by depletion of stratospheric ozone, will change the effectiveness of solar radiation in damaging DNA in human skin. Evaluation of the magnitude of such effects requires knowledge of the altered sunlight spectrum and of the action spectrum for damaging DNA in human skin. We have determined an action spectrum for the frequency of pyrimidine dimer formation induced in the DNA of human skin per unit dose of UV incident on the skin surface. The peak of this action spectrum is near 300 nm and decreases rapidly at both longer and shorter wavelengths. The decrease in our action spectrum for wavelengths less than 300 nm is attributed to the absorption of the upper layers of the skin, an in situ effect that is inherently included in our measurements. Convolution of the dimer action spectrum with the solar spectra corresponding to a solar angle of 40 degrees under current levels of stratospheric ozone (0.32-cm O3 layer) and those for 50% ozone depletion (0.16-cm O3 layer), indicate about a 2.5-fold increase in dimer formation. If the action spectrum for DNA damage that results in skin cancer resembles that for dimer induction in skin, our results, combined with epidemiological data, suggest that a 50% decrease in stratospheric ozone would increase the incidence of nonmelanoma skin cancers among white males in Seattle, Washington, by 7.5- to 8-fold, to a higher incidence than is presently seen in the corresponding population of Albuquerque, New Mexico.
Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent.
Authors: Ferdows M S , Barbour A G
Published: May 2006

Using pulsed-field gel electrophoresis we examined the genome of Borrelia burgdorferi, a eubacterium of the spirochete phylum and the agent of Lyme disease. A population of this species' cells was lysed in situ in agarose blocks. An abundant DNA form that behaved as a linear duplex molecule under different electrophoretic conditions was found. The estimated size of the molecule was 950 kilobases. DNA from two other genera of spirochetes did not enter the gel under these conditions. These studies indicate that Borrelia spirochetes, perhaps uniquely among prokaryotic organisms, have linear chromosomes.
Primary structure of four allatostatins: neuropeptide inhibitors of juvenile hormone synthesis.
Authors: Woodhead A P , Stay B , Seidel S L , Khan M A , Tobe S S
Published: May 2006

Four neuropeptides that inhibit juvenile hormone synthesis by the corpora allata have been isolated from brains of the virgin female cockroach Diploptera punctata. These allatostatins are 8-13 amino acids long, are amidated, and show sequence similarity, including a 3-amino acid sequence at the C-terminal end that is common to all four peptides. The peptide sequences are as follows: allatostatin 1, Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2; allatostatin 2, Gly-Asp-Gly-Arg-Leu-Tyr-Ala-Phe-Gly-Leu-NH2; allatostatin 3, Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2; and allatostatin 4, Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2. An in vitro bioassay of the synthesized allatostatins showed greater than 40% inhibition of juvenile hormone synthesis by corpora allata of virgin females with 10(-9) M allatostatin 1, 10(-8) M allatostatins 2 and 4, and 7 X 10(-7) M allatostatin 3. Inhibition by allatostatins 1-4 was reversible. In addition, allatostatin 1 inhibited juvenile hormone synthesis by corpora allata from mated females and last-instar larvae of D. punctata and corpora allata of adult female Periplaneta americana.
Renal epithelial cyst formation and enlargement in vitro: dependence on cAMP.
Authors: Mangoo-Karim R , Uchic M , Lechene C , Grantham J J
Published: May 2006

Cysts, a common abnormality of kidneys, are collections of urine-like fluid enclosed by a continuous layer of epithelial cells. Renal cysts derive from nephrons and collecting ducts and progressively enlarge as a consequence of epithelial proliferation and transepithelial fluid secretion. The initiation of cyst formation and the factors that control cyst enlargement are unknown. We used an in vitro model of renal cysts to explore the role of the cAMP signal transduction system in the formation and expansion of cysts. MDCK cells, cultured in hydrated-collagen gel, produced polarized monolayered epithelial cysts when intracellular cAMP was increased by prostaglandin E1, arginine vasopressin, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate. All agonists were potentiated by 3-isobutyl-1-methylxanthine, a nucleotide phosphodiesterase inhibitor. The cell proliferation component of cyst enlargement was accelerated by cAMP agonists, as shown by the increased growth of MDCK cells in subconfluent monolayers. The fluid secretion component, reflected by the transepithelial movement of fluid across polarized monolayers of MDCK cells grown on permeable supports, was stimulated by cAMP agonists in the basolateral medium. Chloride levels were higher in the cyst fluid and the secreted fluid than in the bathing medium. We conclude that the development of MDCK cysts is dependent on cAMP. This signal transduction system may be an important modulator of epithelial cell proliferation and transepithelial fluid secretion in the kidney.
High-order fluorescence fluctuation analysis of model protein clusters.
Authors: Palmer A G , Thompson N L
Published: May 2006

The technique of high-order fluorescence fluctuation autocorrelation for detecting and characterizing protein oligomers was applied to solutions containing two fluorescent proteins in which the more fluorescent proteins were analogues for clusters of the less fluorescent ones. The results show that the model protein clusters can be detected for average numbers of observed subunits (free monomers plus monomers in oligomers) equal to 10-100 and for relative fluorescent yields that correspond to oligomers as small as trimers. High-order fluorescent fluctuation analysis may therefore be applicable to cell surface receptor clusters in natural or model membranes.
Date of the monocot-dicot divergence estimated from chloroplast DNA sequence data.
Authors: Wolfe K H , Gouy M , Yang Y W , Sharp P M , Li W H
Published: May 2006

The divergence between monocots and dicots represents a major event in higher plant evolution, yet the date of its occurrence remains unknown because of the scarcity of relevant fossils. We have estimated this date by reconstructing phylogenetic trees from chloroplast DNA sequences, using two independent approaches: the rate of synonymous nucleotide substitution was calibrated from the divergence of maize, wheat, and rice, whereas the rate of nonsynonymous substitution was calibrated from the divergence of angiosperms and bryophytes. Both methods lead to an estimate of the monocot-dicot divergence at 200 million years (Myr) ago (with an uncertainty of about 40 Myr). This estimate is also supported by analyses of the nuclear genes encoding large and small subunit ribosomal RNAs. These results imply that the angiosperm lineage emerged in Jurassic-Triassic time, which considerably predates its appearance in the fossil record (approximately 120 Myr ago). We estimate the divergence between cycads and angiosperms to be approximately 340 Myr, which can be taken as an upper bound for the age of angiosperms.
Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.
Authors: Saiki R K , Walsh P S , Levenson C H , Erlich H A
Published: May 2006

The analysis of DNA for the presence of particular mutations or polymorphisms can be readily accomplished by differential hybridization with sequence-specific oligonucleotide probes. The in vitro DNA amplification technique, the polymerase chain reaction (PCR), has facilitated the use of these probes by greatly increasing the number of copies of target DNA in the sample prior to hybridization. In a conventional assay with immobilized PCR product and labeled oligonucleotide probes, each probe requires a separate hybridization. Here we describe a method by which one can simultaneously screen a sample for all known allelic variants at an amplified locus. In this format, the oligonucleotides are given homopolymer tails with terminal deoxyribonucleotidyltransferase, spotted onto a nylon membrane, and covalently bound by UV irradiation. Due to their long length, the tails are preferentially bound to the nylon, leaving the oligonucleotide probe free to hybridize. The target segment of the DNA sample to be tested is PCR-amplified with biotinylated primers and then hybridized to the membrane containing the immobilized oligonucleotides under stringent conditions. Hybridization is detected nonradioactively by binding of streptavidin-horseradish peroxidase to the biotinylated DNA, followed by a simple colorimetric reaction. This technique has been applied to HLA-DQA genotyping (six types) and to the detection of Mediterranean beta-thalassemia mutations (nine alleles).
A third T-cell receptor beta-chain variable region gene encodes reactivity to Mls-1a gene products.
Authors: Happ M P , Woodland D L , Palmer E
Published: May 2006

Using a quantitative RNA hybridization method, we have identified a third murine T-cell receptor beta-chain variable (V) region gene, V beta 9, that engenders recognition of Mls-1a gene products. V beta 9-expressing T cells are clonally deleted from the periphery of mice that carry the Mls-1a allele, and the fine specificity of V beta 9+ hybridomas suggests that V beta 6-, V beta 8.1-, and V beta 9-encoded T-cell receptors may recognize a similar antigenic epitope. Comparison of these three protein sequences identifies two residues that may be important in determining this recognition specificity.
Neural cell adhesion molecule (N-CAM) is elevated in adult avian slow muscle fibers with multiple terminals.
Authors: Bleisch W , Scharff C , Nottebohm F
Published: May 2006

Many adult avian muscles contain two types of muscle fiber: those that receive innervation at single focal terminals and those with multiple terminals. The muscles of the syrinx, the vocal organ of birds, are such mixed muscles. To study this heterogeneity of fiber type and innervation, we combined immunocytochemistry to classify muscle fibers with techniques to visualize neuromuscular junctions. One monoclonal antibody, S58, directed against a slow class of myosin, labels only fibers that have multiple terminals. We also examined the distribution of immunoreactivity for neural cell adhesion molecule (N-CAM), which has been suggested to play a role in innervation of muscle and formation of neuromuscular junctions. S58-positive fibers have elevated N-CAM staining, indicating that multiple innervation of a fiber is correlated with the fiber's expression of high levels of N-CAM immunoreactivity. Most, and perhaps all, fibers that have multiple terminals also contain abundant N-CAM immunoreactivity. This suggests that N-CAM may play a role in the maintenance of multiterminal innervation in adult innervated muscle.
Age-related development of a heterozygous phenotype in solitary neurons of the homozygous Brattleboro rat.
Authors: van Leeuwen F , van der Beek E , Seger M , Burbach P , Ivell R
Published: May 2006

A single-base deletion in the single-copy vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). It results in the synthesis of an altered vasopressin precursor of which the axonal transport is blocked. Paradoxically, a small number of solitary hypothalamic neurons displays all the immunoreactivities of the wild-type vasopressin precursor (i.e., vasopressin, neurophysin, and a glycopeptide). In the present paper we provide evidence that these neurons have undergone a switch to a genuine heterozygous (di/+) phenotype; i.e., they contain the immunoreactivities of both the wild-type and the mutated vasopressin precursors. In the neural lobe, glycopeptide fibers are also present, showing that axonal transport of the wild-type precursor is restored. Moreover, the number of neurons displaying this di/+ phenotype increases markedly and in a linear way (from 0.1% up to 3% of the vasopressin cells) with age. These findings indicate that after mitotic division has ceased, genomic alterations occur in somatic neurons in vivo. The molecular event generating the di/+ phenotype in the di/di animal could involve a somatic intrachromosomal gene conversion between the homologous exons of the vasopressin and the related oxytocin genes.
Selective expression of a protein-tyrosine kinase, p56lyn, in hematopoietic cells and association with production of human T-cell lymphotropic virus type I.
Authors: Yamanashi Y , Mori S , Yoshida M , Kishimoto T , Inoue K , Yamamoto T , Toyoshima K
Published: May 2006

This paper reports the identification of the lyn gene product, a member of the src-related family of protein-tyrosine kinases, and its expression in hematopoietic cells. A lyn-specific sequence (Arg-25 to Ala-119 of the protein) was expressed in Escherichia coli as a fusion protein with beta-galactosidase. Antiserum raised against the fusion protein immunoprecipitated a 56-kDa protein from human B lymphocytes. Incubation of the immunoprecipitate with [gamma-32P]ATP resulted in the phosphorylation of this protein at tyrosine residues. Immunohistological and immunoblotting analyses showed that the lyn gene product was expressed in lymphatic tissues (spleen and tonsil) and in adult lung, which contains many macrophages. Furthermore, both the transcripts and the protein products of the lyn gene accumulated in macrophages/monocytes, platelets, and B lymphocytes but were not expressed appreciably in granulocytes, erythrocytes, or T lymphocytes, suggesting that lyn gene products function primarily in certain differentiated cells of lymphoid and myeloid lineages.
Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles
Authors: Klein Theodore M. , Roth Bradley A. , Fromm Michael E.
Published: May 2006

We have employed microprojectiles to deliver genes involved in anthocyanin biosynthesis to cells within intact aleurone and embryo tissues of maize. Clones of the
A1
or
Bz1
genes were introduced into aleurone tissue that lacked anthocyanins due to mutations of the endogenous
A1
or
Bz1
gene. Following bombardment, cells within the aleurone developed purple pigmentation, indicating that the mutation in the
a1
or
bz1
genotypes was corrected by the introduced gene. To analyze the expression of these genes in different genetic backgrounds, chimeric genes containing the 5′ and 3′ regions of the
A1
or
Bz1
genes fused to a luciferase coding region were constructed. These constructs were introduced into aleurones of genotypes carrying either dominant or recessive alleles of the
C1
and
R
genes, which are known to regulate anthocyanin production. Levels of luciferase activity in permissive backgrounds (
C1, R
) were 30- to 200-fold greater than those detected in tissue carrying one or both of the recessive alleles (
c1, r
) of these genes. These results show that genes delivered to intact tissues by microprojectiles are regulated in a manner similar to the endogenous genes. The transfer of genes directly to intact tissues provides a rapid means for analyzing the genetic and tissue-specific regulation of gene expression.