bluuebunny/crossref_metadata_2025 huggingface dataset:
Allelic sequence variations in the hypervariable region of a T-cell receptor beta chain: correlation with restriction fragment length polymorphism in human families and populations.
Authors: Robinson M A
Published: May 2006

Direct sequence analysis of the human T-cell antigen receptor (TCR) V beta 1 variable gene identified a single base-pair allelic variation (C/G) located within the coding region. This change results in substitution of a histidine (CAC) for a glutamine (CAG) at position 48 of the TCR beta chain, a position predicted to be in the TCR antigen binding site. The V beta 1 polymorphism was found by DNA sequence analysis of V beta 1 genes from seven unrelated individuals; V beta 1 genes were amplified by the polymerase chain reaction, the amplified fragments were cloned into M13 phage vectors, and sequences were determined. To determine the inheritance patterns of the V beta 1 substitution and to test correlation with V beta 1 restriction fragment length polymorphism detected with Pvu II and Taq I, allele-specific oligonucleotides were constructed and used to characterize amplified DNA samples. Seventy unrelated individuals and six families were tested for both restriction fragment length polymorphism and for the V beta 1 substitution. The correlation was also tested using amplified, size-selected, Pvu II- and Taq I-digested DNA samples from heterozygotes. Pvu II allele 1 (61/70) and Taq I allele 1 (66/70) were found to be correlated with the substitution giving rise to a histidine at position 48. Because there are exceptions to the correlation, the use of specific probes to characterize allelic forms of TCR variable genes will provide important tools for studies of basic TCR genetics and disease associations.
Two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus that were derived from nasopharyngeal carcinomas.
Authors: Glaser R , Zhang H Y , Yao K T , Zhu H C , Wang F X , Li G Y , Wen D S , Li Y P
Published: May 2006

Two epithelial tumor cell lines were established from biopsy specimens of nasopharyngeal carcinomas (NPC). The specimens were taken from poorly differentiated squamous cell carcinomas of the nasopharynx. The tissues were prepared for cell culture and eventually two continuous epithelial cell lines were obtained and designated HONE-1 and HNE-1. Light and electron microscopic examination of these two cell lines demonstrated cells with an epithelial morphology including the presence of desmosomes. The HNE-1 cell line has been passaged more than 100 times and the HONE-1 cell line has been passaged more than 90 times. It was found that early-passage uncloned HNE-1 cells (passage 23) could be superinfected with the B95-8 and NPC-EBV isolates as demonstrated by the induction of Epstein-Barr virus (EBV)-specific early antigen(s) in a small percentage of the cells; HONE-1 cells could also be superinfected with EBV. Southern blot analysis detected EBV DNA in samples from uncloned HNE-1 cells at passages 12, 17, 21, 27, and 35. However, by passage 45, EBV DNA could no longer be detected in HNE-1 cells by Southern blot analysis. The EBV genome was detected in parental HONE-1 cells at subculture 9 and in clone 40 cells up to passage 40 thus far. When HNE-1 cells were examined for the expression of the EBV-encoded nuclear antigen (EBNA) at passage 12, only about 10% of the cells were found to be positive. The percentage of EBNA-positive HNE-1 cells decreased as the cells were passaged. A similar loss of EBNA was observed in uncloned HONE-1 cells, but not in HONE-1 clone 40 cells. In clone 40, which has been passaged 40 times thus far, 85-90% of the cells are still EBNA-positive. The data suggest that EBV genome-positive HNE-1 and HONE-1 cells were lost as the cells were cultivated in vitro and that cloning the cells at an early passage level may be critical in maintaining EBV genome-positive epithelial NPC cells. These EBV genome-positive epithelial NPC cell lines will be useful for studying the association of EBV and NPC.
Localization of mRNA for neural cell adhesion molecule (N-CAM) polypeptides in neural and nonneural tissues by in situ hybridization.
Authors: Prieto A L , Crossin K L , Cunningham B A , Edelman G M
Published: May 2006

The differential expression of the mRNA for the neural cell adhesion molecule (N-CAM) has been studied by in situ hybridization and compared with protein localization analyzed by immunohistochemical staining. The distribution of mRNA during chicken embryonic development was analyzed in neural and nonneural tissues by using an RNA probe that detects all N-CAM mRNAs and a probe specific for the mRNA of the large cytoplasmic domain (ld) of N-CAM. The results provide a detailed description of the mRNA distribution for N-CAM. The distribution of mRNA for total N-CAM generally corresponded to that of protein but differed at a more detailed level of analysis. For example, the mRNA was localized only within the cell bodies of neurons, whereas the protein was also in neuronal processes; this differential localization was most clearly seen in the alternating layers of cell bodies and fibers in the optic tectum and cerebellum. N-CAM ld mRNA, which arises from alternative RNA splicing, was expressed only in neural tissues, confirming previous biochemical and histological studies. Differential expression of the ld mRNA was detected in specific neural cell types: N-CAM mRNA was present in the ependymal cells of the spinal cord and optic tectum, but mRNA for the ld form was absent. In contrast, the ld mRNA was among the N-CAM mRNAs found in the Purkinje cells and internal granule cells in the cerebellum. The differential expression of mRNAs for the N-CAM forms emphasizes the potential importance of alternative mRNA splicing in modulating adhesive events during embryonic development, particularly in the nervous system.
Costimulatory signal provided by a B-lymphoblastoid cell line and its Ia-negative variant.
Authors: Reiser H , Benacerraf B
Published: May 2006

We have analyzed the requirements of highly purified, resting murine CD4+ T lymphocytes for activation mediated by the lectin Con A and by monoclonal antibodies against the CD3 and Thy-1 molecules. Our results indicate that both the Ia-positive B-lymphoblastoid cell line M12 and its Ia-negative variant M12.C3 can provide the costimulatory activity necessary for these activation pathways. The costimulatory function is preserved upon fixation with paraformaldehyde, indicating that the costimulatory molecule(s) is (are) constitutively expressed on the cell surface. Our experiments also point to interesting differences between the M12 cell line and syngeneic Ia-positive antigen-presenting cells in generating a syngeneic mixed lymphocyte reaction. Finally, we show that the CD4+ T cell-M12.C3 cell interaction can be used to screen for interesting monoclonal antibodies that affect cell function.
Photoregulation of a phytochrome gene promoter from oat transferred into rice by particle bombardment.
Authors: Bruce W B , Christensen A H , Klein T , Fromm M , Quail P H
Published: May 2006

The regulatory photoreceptor phytochrome controls the transcription of its own phy genes in a negative feedback fashion. We have exploited microprojectile-mediated gene transfer to develop a rapid transient expression assay system for the study of DNA sequences involved in the phytochrome-regulated expression of these genes. The 5'-flanking sequence and part of the structural region of an oat phy gene have been fused to a reporter coding sequence (chloramphenicol acetyltransferase, CAT) and introduced into intact darkgrown seedlings by using high-velocity microprojectiles. Expression is assayable in less than 24 hr from bombardment. The introduced oat phy-CAT fusion gene is expressed and down-regulated by white light in barley, rice, and oat, whereas no expression is detected in three dicots tested, tobacco, cucumber, and Arabidopsis thaliana. In bombarded rice shoots, red/far-red light-reversible repression of expression of the heterologous oat phy-CAT gene shows that it is regulated by phytochrome in a manner parallel to that of the endogenous rice phy genes. These data indicate that the transduction pathway components and promoter sequences involved in autoregulation of phy expression have been evolutionarily conserved between oat and rice. The experiments show the feasibility of using high-velocity microprojectile-mediated gene transfer for the rapid analysis of light-controlled monocot gene promoters in monocot tissues that until now have been recalcitrant to such studies.
Binding of the herpes simplex virus major regulatory protein to viral DNA.
Authors: Michael N , Roizman B
Published: May 2006

Infected-cell protein 4 (ICP4), the major regulatory protein specified by herpes simplex virus 1 in infected cells, binds to homologs of the sequence ATCGTCnnnnYCGRC (A sites, where n is any nucleotide, Y is a pyrimidine, and R is a purine) and to unrelated sequences for which no consensus sequence has been derived (B sites). We have examined the binding of ICP4 to each of two A and two B binding sites by using Fab fragments of a monoclonal antibody that is reactive with an epitope located at the N terminus of ICP4 and that decreases the mobility of ICP4-DNA complexes in non-denaturing gels. The results indicate that each type of site binds two monomers of ICP4. Methylation-interference studies on the type B sites mapped the guanines whose methylation interfered with the binding of ICP4. The methylation-interference pattern obtained with one of the B sites was similar to that obtained on an A site but differed from that of the other B site. The ability of ICP4 to bind to DNA fragments containing the binding site appears to be dependent on length and on the proximity of the binding site to the fragment end. Short DNA fragments did not form stable complexes with ICP4 even though they contained all of the purines whose methylation interfered with the binding of the regulatory protein.
Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1.
Authors: Bender A , Pringle J R
Published: May 2006

Genes CDC24, CDC42, and CDC43 are required for the establishment of cell polarity and the localization of secretion in Saccharomyces cerevisiae; mutants defective in these genes fail to form buds and display isotropic expansion of the cell surface. To identify other genes that may be involved in these processes, we screened yeast genomic DNA libraries for heterologous genes that, when overexpressed from a plasmid, can suppress a temperature-sensitive cdc24 mutation. We identified four such genes. One of these proved to be CDC42, which has previously been shown to be a member of the rho (ras-homologous) family of genes, and a second is a newly identified ras-related gene that we named RSR1. RSR1 maps between CDC62 and ADE3 on the right arm of chromosome VII; its predicted product is approximately 50% identical to other proteins in the ras family. Deletion of RSR1 is nonlethal but disrupts the normal pattern of bud site selection. Although both CDC42 and RSR1 can suppress cdc24 and both appear to encode GTP-binding proteins, these genes do not themselves appear to be functionally interchangeable. However, one of the other genes that was isolated by virtue of its ability to suppress cdc24 can also suppress cdc42. This gene, named MSB1, maps between ADE9 and HIS3 on the right arm of chromosome XV.
Host species-specific conservation of a family of repeated DNA sequences in the genome of a fungal plant pathogen.
Authors: Hamer J E , Farrall L , Orbach M J , Valent B , Chumley F G
Published: May 2006

We have identified a family of dispersed repetitive DNA sequences in the genome of Magnaporthe grisea, the fungus that causes rice blast disease. We have named this family of DNA sequences "MGR" for M. grisea repeat. Analysis of five MGR clones demonstrates that MGR sequences are highly polymorphic. The segregation of MGR sequences in genetic crosses and hybridization of MGR probes to separated, chromosome-size DNA molecules of M. grisea shows that this family of sequences is distributed among the M. grisea chromosomes. MGR sequences also hybridize to discrete poly(A)+ RNAs. Southern blot analysis using a MGR probe can distinguish rice pathogens from various sources. However, MGR sequences are not highly conserved in the genomes of M. grisea field isolates that do not infect rice. These results suggest that host selection for a specific pathogen genotype has occurred during the breeding and cultivation of rice.
Cardiac allograft survival in mice treated with IL-2-PE40.
Authors: Lorberboum-Galski H , Barrett L V , Kirkman R L , Ogata M , Willingham M C , FitzGerald D J , Pastan I
Published: May 2006

IL-2-PE40 is a chimeric protein composed of human interleukin 2 (IL-2) genetically fused to the amino terminus of a modified form of Pseudomonas exotoxin lacking its cell recognition domain. IL-2-PE40, which is extremely cytotoxic to IL-2 receptor-positive cells, was examined for its ability to prevent graft rejection in mice in which activation of T cells is prominent. We demonstrate that intraperitoneally administered IL-2-PE40 specifically and significantly prolongs the survival of vascularized heart allografts in mice. The chimeric toxin, IL-2-PE40, offers an alternative approach to the treatment of autoimmune diseases and transplant rejection in humans.
Process elimination underlies ontogenetic change in the distribution of callosal projection neurons in the postcentral gyrus of the fetal rhesus monkey.
Authors: Chalupa L M , Killackey H P
Published: May 2006

During fetal development, the regional distribution of callosal projection neurons in the rhesus monkey's postcentral gyrus changes from a uniform to a discontinuous pattern. To determine if this developmental change reflects the retraction of transient callosal projections, two different fluorescent tracers were injected into the brain of fetal monkeys of known gestational ages. Fast blue was injected into the entire postcentral gyrus of one hemisphere, whereas a second tracer (rhodamine latex beads or diamidino yellow) was injected into the caudal portion of the postcentral gyrus of the other hemisphere. The rostral portion of the postcentral gyrus (contralateral to the hemisphere injected with fast blue) was subsequently examined for the presence of labeled cells. In animals injected early in fetal development, on embryonic day 110 or younger and sacrificed 4 weeks later, there were numerous cells labeled with both tracers. In contrast, very few double-labeled cells were found in fetuses injected at an older age, embryonic day 135. We interpret these findings as showing that early in fetal development, when callosal projection neurons in the postcentral gyrus show a continuous distribution pattern, single cells in the rostral portion of this gyrus possess at least two collaterals, one projecting to the contralateral hemisphere and the other to the caudal portion of the gyrus. Subsequently, many of these neurons retract callosal collaterals while maintaining ipsilateral projections. Thus, process elimination accounts for the establishment of the discontinuous distribution of callosal neurons found in the postcentral gyrus of the mature primate.
Alpha-helix and mixed 3(10)/alpha-helix in cocrystallized conformers of Boc-Aib-Val-Aib-Aib-Val-Val-Val-Aib-Val-Aib-OMe.
Authors: Karle I L , Flippen-Anderson J L , Uma K , Balaram H , Balaram P
Published: May 2006

Two molecules of Boc-Aib-Val-Aib-Aib-Val-Val-Val-Aib-Val-Aib-OMe (where Boc is t-butoxycarbonyl and Aib is alpha-aminoisobutyryl) cocrystallize in a triclinic cell with different helical conformations. One molecule is completely alpha-helical with seven 5----1 intramolecular hydrogen bonds. It forms three head-to-tail NH...O = C hydrogen bonds to other molecules of the same conformation. The second molecule has a mixed 3(10)/alpha-helix conformation with three 4----1 hydrogen bonds and four 5----1 hydrogen bonds; furthermore, there is a helix reversal at both termini. The second molecule forms only two head-to-tail hydrogen bonds with molecules of the same type, and the N(3)H group does not participate in any hydrogen bonding. The two different types of helices occur in alternate sheets in the crystal, where each sheet is composed of adjacent rods of helices formed by head-to-tail hydrogen bonding. Within each sheet, containing helices of only one type of conformation, the helices aggregate in a parallel mode. Between the sheets of different helices, the aggregation is antiparallel. The peptide, with formula C51H92N10O13, crystallizes in space group P1 with Z = 2 and cell parameters a = 10.047 +/- 0.002 A, b = 16.684 +/- 0.003 A, c = 19.198 +/- 0.004 A, alpha = 80.30 degrees +/- 0.01 degrees, beta = 85.74 degrees +/- 0.01 degrees, and gamma = 83.03 degrees +/- 0.01 degrees; overall agreement factor R = 6.7% for 6053 data ([F0] greater than 3 sigma) and 0.96-A resolution.
Phosphorylation at threonine-654 is not required for negative regulation of the epidermal growth factor receptor by non-phorbol tumor promoters.
Authors: Friedman B A , van Amsterdam J , Fujiki H , Rosner M R
Published: May 2006

Phosphorylation of the epidermal growth factor (EGF) receptor following activation of protein kinase C appears to negatively regulate EGF binding and the receptor-associated tyrosine kinase activity. We have identified two agents, the calcium ionophore A23187 and the non-phorbol tumor promoter thapsigargin, that similarly inhibit the EGF receptor binding and kinase activities through protein kinase C-independent pathways. Both agents activate protein kinases that phosphorylate the EGF receptor in A431 cells. To test the hypothesis that negative regulation of the EGF receptor always occurs through phosphorylation of threonine-654, a site uniquely phosphorylated by protein kinase C, we analyzed the tryptic phosphopeptides of EGF receptors isolated from cells treated with these agents. While limited phosphorylation of threonine-654 results from the A23187 treatment, no significant phosphorylation of this residue is detected after thapsigargin treatment. These results suggest that EGF receptor phosphorylation is a general mechanism for altering receptor properties and that site(s) of phosphorylation other than threonine-654 may negatively regulate the kinase activity as well as the binding of the EGF receptor.
Identification of resonances from an oncogenic activating locus of human N-RAS-encoded p21 protein using isotope-edited NMR.
Authors: Burk S C , Papastavros M Z , McCormick F , Redfield A G
Published: May 2006

A sample of Escherichia coli-expressed human N-RAS-encoded p21, a 21-kDa protein, was selectively labeled with 15N at each of the 14 glycine amide positions. Two-dimensional proton-observe 15N correlation spectra showed one peak for each glycine residue. Five glycine resonances were identified with residues near the nucleotide binding site and provide useful reporters of several oncogene-activating positions. Three of these resonances were assigned to residues 10, 15, and 115 from the spectrum of a sample that was also labeled with [13C]valine. These resonances showed extra splitting or broadening due to the 13C label, which could be eliminated by 13C decoupling. Two other peaks were unambiguously identified as Gly-12 and Gly-13 using a one-dimensional edited nuclear Overhauser experiment and by spectral comparison with an Asp-12 mutant. These assignments have provided several site-specific probes of critical domains in p21.
Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin.
Authors: Yannas I V , Lee E , Orgill D P , Skrabut E M , Murphy G F
Published: May 2006

Regeneration of the dermis does not occur spontaneously in the adult mammal. The epidermis is regenerated spontaneously provided there is a dermal substrate over which it can migrate. Certain highly porous, crosslinked collagen-glycosaminoglycan copolymers have induced partial morphogenesis of skin when seeded with dermal and epidermal cells and then grafted on standard, full-thickness skin wounds in the adult guinea pig. A mature epidermis and a nearly physiological dermis, which lacked hair follicles but was demonstrably different from scar, were regenerated over areas as large as 16 cm2. These chemical analogs of extracellular matrices were morphogenetically active provided that the average pore diameter ranged between 20 and 125 microns, the resistance to degradation by collagenase exceeded a critical limit, and the density of autologous dermal and epidermal cells inoculated therein was greater than 5 x 10(4) cells per cm2 of wound area. Unseeded copolymers with physical structures that were within these limits delayed the onset of wound contraction by about 10 days but did not eventually prevent it. Seeded copolymers not only delayed contraction but eventually arrested and reversed it while new skin was being regenerated. The data identify a model extracellular matrix that acts as if it were an insoluble growth factor with narrowly specified physiochemical structure, functioning as a transient basal lamina during morphogenesis of skin.
Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase.
Authors: McGuire M C , Nogueira C P , Bartels C F , Lightstone H , Hajra A , Van der Spek A F , Lockridge O , La Du B N
Published: May 2006

A point mutation in the gene for human serum cholinesterase was identified that changes Asp-70 to Gly in the atypical form of serum cholinesterase. The mutation in nucleotide 209, which changes codon 70 from GAT to GGT, was found by sequencing a genomic clone and sequencing selected regions of DNA amplified by the polymerase chain reaction. The entire coding sequences for usual and atypical cholinesterases were compared, and no other consistent base differences were found. A polymorphic site near the C terminus of the coded region was detected, but neither allele at this locus segregated consistently with the atypical trait. The nucleotide-209 mutation was detected in all five atypical cholinesterase families examined. There was complete concordance between this mutation and serum cholinesterase phenotypes for all 14 heterozygous and 6 homozygous atypical subjects tested. The mutation causes the loss of a Sau3A1 restriction site; the resulting DNA fragment length polymorphism was verified by electrophoresis of 32P-labeled DNA restriction fragments from usual and atypical subjects. Dot-blot hybridization analysis with a 19-mer allele-specific probe to the DNA amplified by the polymerase chain reaction distinguished between the usual and atypical genotypes. We conclude that the Asp-70----Gly mutation (acidic to neutral amino acid substitution) accounts for reduced affinity of atypical cholinesterase for choline esters and that Asp-70 must be an important component of the anionic site. Heterogeneity in atypical alleles may exist, but the Asp-70 point mutation may represent an appreciable portion of the atypical gene pool.
PrlA is important for the translocation of exported proteins across the cytoplasmic membrane of Escherichia coli.
Authors: Bieker K L , Silhavy T J
Published: May 2006

Strains of Escherichia coli in which lacZ (specifies beta-galactosidase) is fused to genes that specify exported proteins such as LamB (lambda receptor) exhibit unusual phenotypes. In particular, such strains are killed by high-level expression of the LacZ hybrid protein. Previous results suggest that this overproduction phenotype is the consequence of a lethal jamming of the cellular protein export machinery and this hypothesis is supported by the observed accumulation of the precursor forms of many noncytoplasmic proteins within the moribund cell. Under conditions in which protein export is compromised, biochemical and immunocytochemical analyses indicate that these hybrid proteins can be found in transmembrane orientation. To identify the cellular component rendered rate-limiting by the LacZ hybrid protein under jamming conditions we have utilized signal sequence mutations, which block entry of the hybrid protein into the export pathway, and a dominant suppressor of these lesions, prlA4. Data obtained with a series of merodiploids heterozygous and homozygous for prlA+ and prlA4 show that PrlA is the component sequestered by hybrid jamming. Taken together, these results suggest that PrlA is a component of the export machinery that functions in the translocation of proteins across the cytoplasmic membrane.
In vitro reconstitution of osmoregulated expression of proU of Escherichia coli.
Authors: Ramirez R M , Prince W S , Bremer E , Villarejo M
Published: May 2006

Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an osmotically inducible, high-affinity transport system for the osmoprotectant glycine betaine in Escherichia coli. Previously, a proU-lacZ fusion gene had been cloned, resulting in plasmid pOS3. In vivo osmoregulation of this extrachromosomal proU-lacZ fusion gene at low copy number showed that the plasmid-encoded fusion contained all the necessary sequences in cis for correctly receiving osmoregulatory signals during induction by osmotic stress and repression by glycine betaine. Using a cell-free (S-30) extract, plasmid pOS3 was then used to program protein synthesis in vitro. The ionic compound potassium glutamate specifically stimulated proU-lacZ expression in a concentration-dependent manner. Potassium acetate also induced some proU expression, but other salts were ineffective, thereby ruling out ionic strength as the stimulatory signal. High concentrations of sucrose, trehalose, or glycine betaine did not induce proU expression in vitro either, eliminating osmolarity per se as the stimulus. Reconstitution in a cell-free system rules out osmoregulatory mechanisms that depend on turgor, trans-membrane signaling, or trans-acting regulators synthesized after osmotic upshock.
Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types.
Authors: Band V , Sager R
Published: May 2006

A medium is described that supports long-term growth in culture of human primary mammary tumor cells, of normal epithelial cells from mammoplasty, and of mammary tumor cell lines. Tumor cells are shown to be distinguishable from normal mammary epithelial cells by morphology, by growth requirements, and by two markers: preferential expression of the HMFG-2 epitope on tumor cells and preferential retention in tumor cell mitochondria of the lipophilic fluorescent dye rhodamine 123. Differential fluorescence of HMFG-2 fluorescein-conjugated antibodies can be used as a basis for separation of normal and tumor cells in a fluorescence-activated cell sorter, as can differential retention of rhodamine 123.
Molecular and cellular responses of islets during perturbations of glucose homeostasis determined by in situ hybridization histochemistry.
Authors: Chen L , Komiya I , Inman L , McCorkle K , Alam T , Unger R H
Published: May 2006

We have evaluated in situ hybridization histochemistry as a means of estimating simultaneously the level of prohormone mRNA and the dimensions of rat pancreatic islets. Localization of the 27-mer 32P-labeled oligonucleotide probes for rat proinsulin I, glucagon, and prosomatostatin I corresponded with localization of antibodies to the three hormones. In normal rats subjected to chronic hyperglycemic clamping, the density of the proinsulin mRNA signal increased 54%, islet size and number increased approximately 100%, while proglucagon mRNA signal was reduced 81%. Resection of 50% of the pancreas increased proinsulin mRNA 36% and proglucagon mRNA 500%; islet area doubled and islet number increased 50%. In 150-day-old diabetic ob/ob mice, there was an 18-fold expansion in islet area, a 4-fold increase in islet number, but no increase in insulin gene expression. In insulin-dependent streptozotocin-treated diabetic rats, islet area and number were profoundly reduced; insulin deprivation failed to raise proinsulin mRNA in surviving beta cells above control levels. Proglucagon mRNA was high despite the hyperglycemia but was reduced by insulin within 1 hr, suggesting that insulin regulates glucagon gene expression or is required for its regulation by glucose. In situ hybridization of rat islets provides a valid semiquantitative index of insulin and glucagon biosynthesis and of islet dimensions and reveals that normal but not diabetic islets meet increased insulin demand by increasing both number and biosynthetic activity of beta cells.
Adaptational "crosstalk" and the crucial role of methylation in chemotactic migration by Escherichia coli.
Authors: Hazelbauer G L , Park C , Nowlin D M
Published: May 2006

We investigated roles of methylation in bacterial chemotaxis by characterizing a methyl-accepting transducer protein incapable of methylation because of amino acid substitutions at the modification sites. Mutant Trg protein recognized ligand and generated excitatory signals that affected flagella but was unable to mediate efficient adaptation or net cellular migration in a relevant chemical gradient. Defects caused by lack of methyl-accepting sites on Trg were suppressed by a sufficient cellular content of other transducer molecules with functional methyl-accepting sites. These observations establish directly that methylation is crucial for transducer-mediated chemotaxis and that neither phosphotransfer reactions among the soluble Che proteins nor other interaction among those chemotactic components can effectively fulfill the functions of methylation. Suppression was correlated with adaptational "crosstalk" in which unoccupied methyl-accepting transducers acquired methyl groups, thus apparently substituting effectively for blocked methyl-accepting sites on the transducer. A plausible model for this phenomenon is that increased methylation of unstimulated transducers results from global inhibition of the demethylating enzyme in a cell with a normally active methyltransferase and no available methyl-accepting sites on the stimulated, mutant transducer. Thus methylation can perform its roles in adaptation and gradient sensing even if modification occurs on molecules different from those that recognize the stimulating compound. This observation emphasizes the central role of methylation and the modular nature of the chemosensory system.
In vitro
phosphorylation of plant plasma membrane proteins in response to the proteinase inhibitor inducing factor
Authors: Farmer Edward E. , Pearce Gregory , Ryan Clarence A.
Published: May 2006

A polygalacturonide purified from a tomato leaf pectic polysaccharide that induces the systemic synthesis of proteinase inhibitors in tomato plants enhances the phosphorylation of specific proteins in plasma membrane fractions isolated from tomato and potato leaves. In tomato plasma membranes, two proteins of 34 and 29 kDa show enhanced phosphorylation in response to the polyuronide. In potato plasma membranes, only a protein of 34 kDa exhibited enhanced phosphorylation due to the polyuronide. A noncarbohydrate class of proteinase inhibitor inducing factor, recently identified by workers in this laboratory, resulted in the
in vitro
hyperphosphorylation of a family of proteins of ≈27 kDa. The phosphorylation of specific polypeptides in leaves in response to the same factors that induce the expression of proteinase inhibitor genes suggests that protein kinases may play an important role in the mechanism of signal transduction leading to defense gene expression.
In vitro effects of recombinant interleukin 7 on growth and differentiation of bone marrow pro-B- and pro-T-lymphocyte clones and fetal thymocyte clones.
Authors: Takeda S , Gillis S , Palacios R
Published: May 2006

We have studied the effects of recombinant (r) interleukin 7 (IL-7) on growth and differentiation of marrow pro-B-lymphocyte clones (CB/Bm7, LyD9, LyB9), marrow pro-T-lymphocyte clones (C4-77/3, C4-86/18, C4-95/16), and fetal thymocyte clones (FTH5, FTA2, FTD5) in the presence or absence of the bone marrow stroma clone RP.0.10, which was selected for its ability to promote differentiation of the pro-B clones. rIL-7 alone stimulated some DNA synthesis (measured by [3H]thymidine uptake) but not actual growth (increase in cell number) of the pro-B clones. Antibodies against IL-4 and IL-6 or against receptors for IL-2, IL-3, and IL-5 did not inhibit this effect of rIL-7 on the pro-B clones. rIL-7 alone or in various combinations with other cytokines (from rIL-1 alpha to rIL-6) could not induce differentiation of the pro-B clones into IgM+ B cells regardless of the presence of lipopolysaccharide (LPS). The RP.0.10 marrow stroma cells by themselves do not support the growth of the pro-B clones. However, the pro-B clones grew when cultured with rIL-7 and monolayers of the RP.0.10 stroma cells. While the RP.0.10 stroma cells induced the pro-B clones to differentiate into IgM+ B cells but not T3+ T cells when cultured in the presence of LPS and rIL-3, the B-cell progenitor clones gave rise to significantly higher numbers of IgM+ B cells (up to 63%) and to many more B cells expressing higher levels of surface IgM when cocultured with rIL-7, LPS, and RP.0.10 stroma cells. The pro-B clones also generated IgM+ B cells (up to 20%) when cocultured with RP.0.10 stroma cells and rIL-7 in the absence of LPS. By using culture plates designed for testing requirements for cell-cell contact, we found that cell interactions between the pro-B cell and the marrow stroma cell are essential to induce rearrangement and expression of the immunoglobulin genes in the pro-B clones. Possible mechanisms to account for the remarkable effects of rIL-7 in the presence of RP.0.10 stroma cells on both growth and differentiation of the pro-B clones are discussed. Finally, rIL-7 alone or together with RP.0.10 stroma cells neither supported proliferation nor induced differentiation into T3+ T cells or IgM+ B cells of the marrow pro-T clones or the fetal thymocyte clones. In light of these findings, we postulate that the interaction of the pluripotential stem cell with marrow stroma cells like RP.0.10 and the availability of IL-7 could play a critical role in the commitment to develop along the B-lymphocyte pathway.
Programmed cell death (apoptosis) in pancreatic cancers of hamsters after treatment with analogs of both luteinizing hormone-releasing hormone and somatostatin.
Authors: Szende B , Zalatnai A , Schally A V
Published: May 2006

Female Syrian golden hamsters with N-nitrosobis(2-oxopropyl)amine (BOP)-induced ductal pancreatic cancers were treated with long-acting microcapsular preparations of the 6-D-tryptophan analog of luteinizing hormone-releasing hormone [( D-Trp6]LH-RH), releasing 25 micrograms/day; the somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160), liberating 15 micrograms/day; and the combination of these two peptides. Therapy with analogs was initiated 24 weeks after initial administration of BOP. These treatments resulted in significantly better survival of all animals as compared to BOP controls; body weights of surviving peptide-treated animals were significantly higher than those of the BOP controls. All 15 BOP-control animals had pancreatic cancers. In the group treated with RC-160 four hamsters were free of tumors, whereas therapy with [D-Trp6]LH-RH resulted in seven tumor-free animals, and combination of RC-160 and [D-Trp6]LH-RH resulted in eight tumor-free animals from groups of 15. Only preblastomatous lesions were found in these animals. Average tumor weight of animals in all peptide-treated groups, sacrificed 60 days after beginning the peptide treatment, was significantly lower than that of BOP controls. No significant differences were seen between the various peptide-treated groups. Histologically, analog-treated tumors of hamsters showed striking regressive changes characteristic of programmed cell death (apoptosis). This apoptosis presumably resulted from hormonal effects on tumor cells from prolonged treatment with these analogs of hypothalamic hormones. Our present data confirm the beneficial effect of long-acting microcapsules of [D-Trp6]LH-RH and RC-160 on pancreatic carcinoma and suggest a mode of action for these peptides. The feasibility of applying this treatment with analogs of hypothalamic hormones to human pancreatic carcinoma can be envisioned from these studies.
Inhibition of growth of human mammary tumor cells by potent antagonists of luteinizing hormone-releasing hormone.
Authors: Sharoni Y , Bosin E , Miinster A , Levy J , Schally A V
Published: May 2006

Various studies support the view that analogs of luteinizing hormone-releasing hormone (LH-RH) exert some direct effects on mammary tumor cells. Recently, new LH-RH antagonists [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Hci6,D-Ala10]-LH-RH (SB-29) and [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Cit6,D-Ala10]LH-RH (SB-30), which are devoid of edematogenic effects, were synthesized. In this study, we examined whether these LH-RH antagonists inhibit the proliferation of MDA-MB-231 human mammary tumor cells in culture. [3H]Thymidine incorporation into DNA and cell number were measured. The antagonists induced up to 40% inhibition of [3H]thymidine incorporation in MDA-MB-231 cells. This inhibition was dose-dependent in the 0.3-30 microM range and could be demonstrated after 2 days of incubation in the presence of the peptides. An older antagonist, [Ac-D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]-LH-RH (ORG 30276), had a lesser effect, and the agonist des-Gly10-[D-Ser(tBu)6]LH-RH ethylamide (buserelin) had no effect. The antagonists SB-29 and SB-30 also inhibited the rate of cell growth, as measured by cell number, while the LH-RH agonist buserelin had no significant effect. These results support the concept that these new LH-RH antagonists can directly inhibit the growth of human mammary tumors and thus might be suitable for the treatment of breast cancer.
Molecular model of the G protein alpha subunit based on the crystal structure of the HRAS protein.
Authors: Holbrook S R , Kim S H
Published: May 2006

A structural model of guanine nucleotide-binding regulatory protein alpha subunits (G alpha subunits) is proposed based on the crystal structure of the catalytic domain of the human HRAS protein (p21ras). Because of low overall sequence similarity, structural and functional constraints were used to align the G alpha consensus sequence with that of p21ras. The resulting G alpha model specifies the spatial relationship among the guanine nucleotide-binding site, the binding site of the beta gamma subunit complex, likely regions of effector and receptor interaction, and sites of cholera and pertussis toxin modification. The locations in the model of the experimentally determined sites of proteolytic digestion, point mutation, monoclonal antibody binding, and toxin modification are consistent with and help explain the observed biological activity. Two important findings from our model are (i) the orientation of the G alpha model with respect to the membrane and (ii) the identification of the spatial proximity of the N- and C-terminal regions. Furthermore, by analogy to p21ras, the model assigns specific residues in G alpha required for binding the guanosine (G-box) and phosphates (PO4-box) and identifies residues potentially involved in the conformational switch mechanism (S-box). Specification of these critical regions in the G alpha model suggests guidelines for construction of mutants and chimeric proteins to experimentally test structural and functional hypotheses.
Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.
Authors: Hemdan E S , Zhao Y J , Sulkowski E , Porath J
Published: May 2006

Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins--thioredoxin, ubiquitin, calmodulin, lysozyme, cytochrome c, and myoglobin on immobilized transition metal ions (Co2+, Ni2+, Cu2+, and Zn2+)--allows establishment of the following facets of the histidyl side chain distribution: (i) either interior or surface; (ii) when localized on the surface, accessible or unaccessible for coordination; (iii) single or multiple; (iv) when multiple, either distant or vicinal. Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution).
Ribosomal DNA and Stellate gene copy number variation on the Y chromosome of Drosophila melanogaster.
Authors: Lyckegaard E M , Clark A G
Published: May 2006

Multigene families on the Y chromosome face an unusual array of evolutionary forces. Both ribosomal DNA and Stellate, the two families examined here, have multiple copies of similar sequences on the X and Y chromosomes. Although the rate of sequence divergence on the Y chromosome depends on rates of mutation, gene conversion and exchange with the X chromosome, as well as purifying selection, the regulation of gene copy number may also depend on other pleiotropic functions, such as maintenance of chromosome pairing. Gene copy numbers were estimated for a series of 34 Y chromosome replacement lines using densitometric measurements of slot blots of genomic DNA from adult Drosophila melanogaster. Scans of autoradiographs of the same blots probed with the cloned alcohol dehydrogenase gene, a single copy gene, served as internal standards. Copy numbers span a 6-fold range for ribosomal DNA and a 3-fold range for Stellate DNA. Despite this magnitude of variation, there was no association between copy number and segregation variation of the sex chromosomes.
Hepatitis B virus X gene can transactivate heterologous viral sequences.
Authors: Twu J S , Robinson W S
Published: May 2006

The smallest open reading frame of hepatitis B virus (HBV) has been designated the X gene and its biological function during HBV infection and replication is not known. Experiments described here demonstrate that expression of the HBV X gene in HepG2 cells containing a plasmid with the chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) sequence leads to a marked increase in CAT gene transcription as well as expression of the gene product (CAT). The HIV-1 tatIII gene and the HBV X gene together increased HIV-1 LTR-regulated CAT expression above that observed with either gene alone, suggesting a synergistic effect of the X gene and tat. HBV X gene also stimulated expression of the CAT gene under control of the simian virus 40 enhancer and early promoter but not the visna virus LTR or the human T-cell lymphotropic virus type I (HTLV-I) LTR, indicating that the HBV X gene can transactivate some but not other heterologous viral sequences. Transactivation of the HIV-1 LTR by the HBV X gene varied in different cell lines, suggesting that it may be mediated by a cellular factor(s).
Cloning of the mRNA for the protein that crosslinks to the egg peptide speract.
Authors: Dangott L J , Jordan J E , Bellet R A , Garbers D L
Published: May 2006

An apparent receptor for the egg peptide speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) was identified by covalently coupling a radiolabeled speract analogue to intact spermatozoa and was then purified by DEAE-Sepharose chromatography and preparative gel electrophoresis after solubilization with Lubrol PX. The purified, crosslinked protein was digested with Staphylococcus aureus V8 protease and a resultant peptide, purified from polyacrylamide slab gel slices, was shown to have the amino acid sequence Val-Ser-Ala-Pro-Phe-Asp-Leu-Glu-Ala-Pro-Phe-Ile-Ile-Asp-Gly-Ile. Polyclonal antiserum, generated against a synthetic peptide that corresponded to the above sequence, immunoprecipitated the radiolabeled crosslinked protein and reacted with a Mr 77,000 protein on immunoblots, demonstrating that the sequenced peptide originated from the apparent receptor. A clone containing a 2.5-kilobase insert was subsequently isolated from a sea urchin testis cDNA library that contained DNA sequences encoding an open reading frame of 532 amino acids that included the above peptide sequence. The deduced amino acid sequence suggests that the protein contains a 26-residue amino-terminal signal peptide, a large extracellular domain relatively rich in cysteine (5%) that includes a four-fold repeat of about 115 amino acids, a single membrane-spanning region, and only 12 amino acid residues extending into the cytoplasm. Analysis of total RNA from Strongylocentrotus purpuratus testis by Northern blot revealed a 2.5-kilobase RNA. Preliminary data show the presence of hybridizing RNA of the same apparent size in other sea urchin species, including Arbacia punctulata, which does not respond to speract.
Mammalian cAMP-responsive element can activate transcription in yeast and binds a yeast factor(s) that resembles the mammalian transcription factor ANF.
Authors: Jones R H , Jones N C
Published: May 2006

The human ATF and AP1 transcription factors bind to highly related DNA sequences. Their consensus binding sites differ by a single nucleotide, but this single change is crucial in determining factor binding specificity. We have previously identified an AP1 (yAP1) binding activity in yeast. In this report we identify a yeast ATF (yATF) binding activity whose specificity can be distinguished from that of yAP1 by the same crucial nucleotide that distinguishes binding of human ATF and AP1. The ATF binding site can act as an efficient upstream activating sequence in vivo, suggesting that yATF is a transcriptional activator. The yATF DNA-binding complex is phosphorylated and the binding activity of partially purified yATF can be enhanced in vitro by the addition of protein kinase A, indicating that the phosphorylation state of yATF may be important in determining its ability to bind DNA.
Neonatal induction of a nuclear protein that binds to the c-fos enhancer.
Authors: Levi B Z , Kasik J W , Burke P A , Prywes R , Roeder R G , Appella E , Ozato K
Published: May 2006

The expression of the c-fos gene is transiently induced at birth in most organs in the mouse. To study the basis of this induction we searched for a nuclear factor that binds to the 5' regulatory region of the c-fos gene. Gel mobility shift assays with tissue extracts revealed fast (band I) and slow (band III) migrating bands, which represent factor binding to the c-fos enhancer, termed the serum response element (SRE). Neonatal extracts preferentially elicited band I, with low or undetectable levels of band III, whereas fetal and adult extracts generated predominantly band III, with reduced levels of band I. These results indicate that the SRE-binding activity changes during perinatal development and that the appearance of band I, which coincides with diminution of band III, correlates with neonatal c-fos induction. Methylation interference and competition analyses showed that the neonatal factor (band I) binds to the SRE at a site different from the adult factor (band III). DNA-binding activity of the adult factor, but not the neonatal factor, was sensitive to phosphatase treatment. Furthermore, the adult factor, but not the neonatal factor, shared antigenic specificity with the human serum response factor (SRF) that is expressed in cultured cells irrespective of c-fos gene induction. We conclude that band I in neonates represents a SRE-binding factor that is distinct from the SRF, which may be responsible for the neonatal induction of the c-fos gene. The band III factor was indistinguishable from the SRF in all criteria tested.