bluuebunny/crossref_metadata_2025 huggingface dataset:
Highly selective photoaffinity labeling of mu and delta opioid receptors.
Authors: Garbay-Jaureguiberry C , Robichon A , Daugé V , Rossignol P , Roques B P
Published: May 2006

We report the synthesis and photolabeling properties of two highly selective ligands for mu and delta opioid-binding sites: Tyr-D-Ala-Gly-MePhe (pN3)-Gly-ol (AZ-DAMGE) and Tyr-D-Thr-Gly-Phe (pN3)-Leu-Thr (AZ-DTLET). An irreversible inhibition of the electrically induced contractions of mouse vas deferens is caused by irradiation (at 254 nm) of the muscle strip in the presence of AZ-DTLET (1 nM). This phenomenon is antagonized only at large concentrations (10 microM) of naloxone, in accordance with the well-known lower selectivity of naloxone for delta sites. Competition experiments with [3H]DAMGE and [3H]DTLET on crude rat brain membranes showed that the azido photoprobes display a similar (AZ-DAMGE) and even a better (AZ-DTLET) selectivity than their respective parent compounds DAMGE and DTLET. Up to 25 nM, AZ-DTLET irreversibly and selectively photolabels the delta sites of crude rat brain homogenates. Due to its lower affinity AZ-DAMGE provides similar selective photolabeling of the mu sites but at higher concentrations (approximately equal to 0.3 microM). When [3H]DAMGE and [3H]DTLET were used as ligands for mu and delta binding subtypes, respectively, no important change in binding capacity and affinity of one receptor type was observed after photolabeling of the other.
Mouse c-mos oncogene activation is prevented by upstream sequences.
Authors: Wood T G , McGeady M L , Baroudy B M , Blair D G , Vande Woude G F
Published: May 2006

Although the molecularly cloned mouse c-mos oncogene locus can be efficiently activated by insertion of a retroviral long terminal repeat (LTR) 5' to its coding region, only low-frequency transformation occurs with the LTR element inserted 3' to this region. Analysis of several of the latter transformed cell lines suggested that loss of 2 kilobases (kb) of normal mouse DNA sequences preceding c-mos was required for oncogene activation. The determination of the transforming potential of deletion mutants containing only portions of this region followed by analysis of their nucleotide sequences identified a region termed upstream mouse sequence (UMS) as a cis-acting locus that prevents c-mos activation by a 3' LTR. The UMS region is approximately 1 kb in length and is located 0.8-1.8 kb upstream from the first ATG in the open reading frame of c-mos. Insertion of UMS 5' to the v-mos coding region also prevents 3' LTR enhancement of its transforming activity, but this inhibition is position dependent and functions only when inserted between v-mos and its putative promoter. The results presented here suggest that UMS may function to regulate c-mos proto-oncogene expression and may explain the lack of detectable c-mos transcripts in normal mouse cells.
Identification of a second transforming region in bovine papillomavirus DNA.
Authors: Schiller J T , Vass W C , Lowy D R
Published: May 2006

Bovine papillomavirus type 1 (BPV-1) has been used as a model for studying papillomavirus genetics because BPV-1 virions or BPV-1 genomic viral DNA efficiently induce morphologic transformation of certain cultured cells. Previous studies of BPV-1-induced transformation have found that a cloned 5.4-kilobase (kb) fragment (69T) of the genome is transforming and that a 2.3-kb segment from the 3' end of this fragment is also transforming if activated by a retroviral regulatory element (the long terminal repeat). We now report that 69T contains another transforming segment near its 5' end that can also be activated by a long terminal repeat. Since this second segment does not overlap the 3' transforming segment, we conclude that BPV-1 encodes at least two genes that can independently transform cultured cells. Mutational analysis of the 5' transforming segment suggests that the transforming gene of this segment lies within the E6 open reading frame. The two transforming segments differ in their biological activity in that the E6-containing fragment can transform C127 mouse cells but not NIH3T3 mouse cells, whereas the 3' fragment can transform both cell lines.
Immunization in vitro and production of monoclonal antibodies specific to insoluble and weakly immunogenic proteins.
Authors: Van Ness J , Laemmli U K , Pettijohn D E
Published: May 2006

A procedure is described for immunizing in vitro and stimulating proliferation of specific B-cell lymphocytes. The method is applicable to production of monoclonal antibodies against proteins that are soluble only in denaturing solvents. An induction period is described in which antigen is presented to the B-cell population in the absence of serum. Also, antigen is coupled to mitogenic silica, which allows the effective presentation of both soluble and insoluble antigens. The results indicate hybridomas can be obtained that secrete IgMs directed against highly conserved or weakly immunogenic antigens.
Interferon induces pulmonary cysts in A2G mice.
Authors: Woodrow D , Moss J , Gresser I
Published: May 2006

Suckling A2G mice were treated for the first week of life with either partially purified or electrophoretically pure mouse alpha/beta interferon. All mice subsequently developed multiple large pulmonary cysts. The lesions were never found in interferon-treated Swiss mice and only very infrequently in interferon-treated BALB/c or F1 Swiss/A2G mice. These observations show that a brief exposure of suckling A2G mice to alpha/beta interferon can result in a severe disease that is determined by the mouse genotype.
Microtubule-associated protein 2: monoclonal antibodies demonstrate the selective incorporation of certain epitopes into Alzheimer neurofibrillary tangles.
Authors: Kosik K S , Duffy L K , Dowling M M , Abraham C , McCluskey A , Selkoe D J
Published: May 2006

Neurofibrillary tangles (NFT) are the principal structural alteration of neuronal cell bodies in Alzheimer disease as well as in normal aging of the human brain. While the ultrastructure of these intraneuronal lesions has been extensively studied, the biochemical composition of the fibers comprising the NFT is unknown. We report the production of three monoclonal antibodies against the microtubule-associated protein 2 (MAP-2), one of which intensely labels Alzheimer NFT. All three antibodies specifically recognize MAP-2 on immunoblots and stain brain tissue in a characteristic dendritic pattern. The three antibodies are directed against at least two different antigenic sites on the MAP-2 molecule, and one appears to recognize a phosphorylation site on MAP-2. That only one of the three antibodies immunolabels NFT suggests that the formation of the tangle involves some modification of the MAP-2 molecule. Our findings suggest that one aspect of Alzheimer-type neurofibrillary pathology is an aggregation of MAP-2 or MAP-2 fragments with altered neurofilamentous elements present in NFT. Normal interactive function, which putatively occurs between neurofilaments and MAP-2, may thus be disrupted in Alzheimer disease.
Genes for the alpha and beta subunits of phycocyanin.
Authors: de Lorimier R , Bryant D A , Porter R D , Liu W Y , Jay E , Stevens S E
Published: May 2006

Phycocyanin (PC) is a light-harvesting protein common to blue-green and red algae. We have isolated the genes for the two apoprotein subunits, alpha and beta, of PC from the blue-green alga Agmenellum quadruplicatum PR-6. We synthesized eight sense-strand tetradecameric oligonucleotide probes that could encode a particular pentapeptide in PC alpha from A. quadruplicatum. Only one probe hybridized with total RNA from this organism. This oligonucleotide showed homology to a unique restriction fragment when used to probe Southern blots of A. quadruplicatum DNA. The probe-homologous 3.1-kilobase pair HindIII fragment was cloned. The nucleotide sequence of a 1.7-kilobase pair segment of this clone was determined. Two open reading frames are contained in this region, which correspond in deduced amino acid sequence to PC alpha and PC beta subunits. Both coding sequences are in the same orientation, separated by 105 base pairs, with the PC beta gene 5' to the PC alpha gene. Each gene has a Shine-Dalgarno-type sequence near the initiation codon. Codon frequencies in the two genes may be correlated with the abundance of their products. The deduced amino acid sequences of the gene products show considerable homology with the alpha and beta PC subunits from other species.
Substances originating from the optic nerve of neonatal rabbit induce regeneration-associated response in the injured optic nerve of adult rabbit.
Authors: Hadani M , Harel A , Solomon A , Belkin M , Lavie V , Schwartz M
Published: May 2006

We have recently shown that cell bodies of an injured optic nerve of adult rabbit can be induced to express regeneration-associated response by external signals derived from nonneuronal cells of regenerating nerves of lower vertebrates. In this study it is shown that even substances derived from a nonregenerating mammalian system also can trigger such a regenerative response. Thus, substances derived from intact nerves of neonatal rabbits and of adult rabbits, to a lesser extent, were active in triggering a regeneration-associated response, whereas substances derived from injured nerves of adult rabbit were not. However, if subsequent to the injury the nerve was implanted with silicone tube containing medium conditioned by neonatal optic nerves, the substances derived from the implanted injured nerve were active. Thus, it appears that the ability of a periaxonal environment to provide triggering substances correlates with axonal growth. Therefore, we named these substances "growth-associated triggering factors" (GATFs). It is suggested that mammalian cells are unable to express a regenerative response after an injury due to the failure of their nonneuronal cells to produce regeneration-triggering substances. This disability may be circumvented by an appropriate implantation procedure.
Base (O.-2, e-, or OH-)-induced autoxygenation of organic substrates: a model chemical system for cytochrome P-450-catalyzed monoxygenation and dehydrogenation by dioxygen.
Authors: Sawyer D T , Sugimoto H , Calderwood T S
Published: May 2006

When catalytic quantities of superoxide ion (O.-2; or of electrons from electrolysis or of OH-) are introduced into a dry acetonitrile solution that contains excess substrate (RH), ambient air (O2), 1,2-diphenylhydrazine (PhNHNHPh), and iron(II), the substrate is rapidly and efficiently monoxygenated (e.g., triphenylphosphine----triphenylphosphine oxide, benzyl alcohol----benzaldehyde, diphenylsulfoxide----diphenylsulfone) or dehydrogenated (1,4-cyclohexadiene----benzene). The model consists of (i) an O2-activation segment that produces H2O2 from an O.-2-initiated autoxidation of disubstituted hydrazine (a model for reduced flavin), (formula; see text) and (ii) a H2O2-activation segment via the iron(II)-induced formation of ferryl ion (FeO2+), Fe(II) + H2O2----FeO2 + H2O, an effective monoxygenating agent: FeO2+ + RH----Fe(II) + ROH. The combination of i and ii provides a catalytic system for the autoxidation of organic substrates with reaction cycles that are similar to those for cytochrome P-450 monoxygenases.
Estimation of continental
187
Os/
186
Os values by using
187
Os/
186
Os and
143
Nd/
144
Nd ratios in marine manganese nodules
Authors: Turekian Karl K. , Luck Jean-Marc
Published: May 2006

The relationship between
187
Os/
186
Os and
143
Nd/
144
Nd in different manganese nodule fields is used to determine the
187
Os/
186
Os ratio of the continental terrains bounding the major ocean basins. The Atlantic Ocean drainages yield
187
Os/
186
Os of about 11; the Pacific Ocean, between 25 and 36; and the western Indian Ocean, 20. By assuming a two-component continental crust composed of “ultramafic rocks” (high Os concentration, low
187
Os/
186
Os) and “granite” with only radiogenic
187
Os produced in accessory Re-bearing molybdenite, the ultramafic contribution to weathering is about 0.2%. Some or most of this may come from the alteration of oceanic ultramafics.
Quantitation of aflatoxin B1 adduction within the ribosomal RNA gene sequences of rat liver DNA.
Authors: Irvin T R , Wogan G N
Published: May 2006

The in vivo formation of covalent aflatoxin B1 (AFB1)-DNA adducts within the rRNA gene sequences of nuclear DNA has been studied in AFB1-treated rats. Liver nuclear DNA, enriched in ribosomal DNA (rDNA) by one round of cesium salt density gradient centrifugation, was treated under buffered alkaline conditions to convert unstable AFB1-N7-guanine adducts to stable AFB1-formamidopyrimidine derivatives. The alkali-treated DNA was hybridized to 18S and 28S rRNA in 70% formamide buffer to form rRNA X rDNA hybrids. These hybrids were separated from the bulk of nuclear DNA by two rounds of centrifugation in CsCl, and the level of AFB1 adduction to rDNA versus total nuclear DNA was compared as a function of dose 2 hr after AFB1 administration. Over an 8-fold dose range (0.25-2.0 mg of AFB1 per kg of body weight), rDNA contained 4- to 5-fold more AFB1 residues than nuclear DNA, indicating that rDNA is preferentially accessible to carcinogen modification in vivo. While aflatoxin B1 forms adducts with DNA principally at guanine residues, the guanine enrichment of rDNA was insufficient to explain the magnitude of observed preferential AFB1 modification of rDNA. These results support the hypothesis that rDNA regions are preferentially accessible to carcinogen modification because of the diffuse conformation maintained within transcribed genes. This experimental approach permits the quantitative description of carcinogen modification within a defined gene sequence; further refinement of this approach may be useful in defining the precise relationships between covalent chemical-DNA interactions and the alterations in gene expression that result.
Human pancreatic icosapeptide: isolation, sequence, and immunocytochemical localization of the COOH-terminal fragment of the pancreatic polypeptide precursor.
Authors: Schwartz T W , Hansen H F , Håkanson R , Sundler F , Tager H S
Published: May 2006

In dogs, the COOH-terminal part of the pancreatic polypeptide precursor gives rise to a stable icosapeptide product against which an antiserum has been raised. By immunohistochemistry, icosapeptide immunoreactivity was localized in human pancrease exclusively to cells that also stored pancreatic polypeptide. Analytical peptide chemistry demonstrated that a peptide corresponding to the canine icosapeptide could be extracted from the pancreatic polypeptide-rich duodenal part of the human pancreas. The human pancreatic icosapeptide was isolated by acid ethanol extraction, gel filtration, anion-exchange chromatography, and reverse-phase high-performance liquid chromatography. The COOH-terminal sequence of the human icosapeptide is very similar to that of the canine icosapeptide, whereas none of the first nine amino acid residues are identical. When the primary structure of peptides from three different species are compared, it is apparent that the pancreatic polypeptide part of the common precursor is a well-conserved sequence as compared to the icosapeptide part, although 8 out of 11 residues in the COOH-terminal sequence of the icosapeptide are identical in all three species.
Sensitive light scattering probe of enzymatic processes in retinal rod photoreceptor membranes
Authors: Lewis J. W. , Miller J. L. , Mendel-Hartvig J. , Schaechter L. E. , Kliger D. S. , Dratz E. A.
Published: May 2006

Light excitation of as little as 0.05% of the rhodopsin in a retinal rod membrane suspension reduces the near-IR optical transmission by 25%. This transmission decrease requires the presence of guanosine triphosphate, is opposite in sign and 25 times larger in amplitude than a GTP-dependent light-scattering signal previously reported in rod outer segment suspensions [Kuhn, H., Bennett, N., Michel-Vallez, M. & Chabre, M. (1981)
Proc. Natl. Acad. Sci. USA
, 78, 6873-6877], and is kinetically complex. The initial phase of the optical transmission decrease begins after about a 50-ms lag (at 0.05% bleach) and has a first-order time constant of 300-500 ms. The scattering signal returns to the preactinic baseline in a time dependent on the amount of GTP added. A nonhydrolyzable GTP analogue, guanylyl imidodiphosphate, produces a scattering signal that does not return to the preactinic baseline. Adenosine triphosphate strongly inhibits the return of the GTP-dependent transmission decrease to the preactinic baseline. This effect of ATP on the GTP signal apparently requires ATP hydrolysis because it is inhibited by the simultaneous presence of adenylyl imidodiphosphate, a nonhydrolyzable analogue of ATP. The light-scattering signal and the velocity of the activation of a rod outer segment phosphodiesterase saturate when >0.05% of the rhodopsin is bleached and both show nearly identical dependence on light stimulus. It is suggested that these nucleotide-dependent light-scattering signals arise from changes in the state of membrane aggregation that are controlled by enzymatic processes. This hypothesis is supported by the large amplitude of the signals, sedimentation experiments, and a strong membrane concentration dependence. The ATP effects can be rationalized within the above hypothesis as being due to ATP-dependent rhodopsin phosphorylation that adds negative charges to the membrane surface and tends to keep the membranes disaggregated. An additional signal, which increases light transmission, is produced by a second, much more intense flash. The latter signal is interpreted as the result of proton binding by bleached rhodopsin molecules that decreases the negative charge repulsion between the membranes and allows increased aggregation.
Isolation and partial renaturation of proteolytic fragments of the myosin head.
Authors: Muhlrad A , Morales M F
Published: May 2006

Methods have been devised for isolating two of the tryptic fragments (those termed "20K" and "50K") of myosin "subfragment 1" in pure form. Fragment 20K was examined for renaturation after removal of denaturants used in its preparation. It generated a CD spectrum corresponding to ca. 64% formed structure (roughly what would be expected from its amino acid sequence) and a red-shifted UV spectrum such as arises when phenylalanine and tyrosine are perturbed by structural interactions. Actin affinity of fragment 20K was tested by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking, inhibition of the actin-activated ATPase of subfragment 1 containing light chain 3, cosedimentation with actin, and light scattering; the affinity exceeded 5 X 10(6) M-1. The foregoing suggests that moiety 20K has a sovereign existence in (i.e., is a domain of) myosin subfragment 1. Preliminary work indicates that fragment 50K also binds actin, but with a lesser affinity.
Biologic activity in a fragment of recombinant human interferon alpha.
Authors: Ackerman S K , Zur Nedden D , Heintzelman M , Hunkapiller M , Zoon K
Published: May 2006

To attempt to locate functionally important regions of the interferon (IFN) molecule, recombinant human IFN-alpha 2 was subjected to proteolytic digestion. The bacterial proteinase thermolysin produced two major complementary fragments, HuIFN-alpha 2-(1-110) and HuIFN-alpha 2-(111-153). After reduction with 2-mercaptoethanol and separation of the two major fragments on NaDodSO4/polyacrylamide gel electrophoresis, antiviral activity persisted in the larger, Mr 12,000, fragment consisting of the amino-terminal 110 amino acids.
Structure of a human gastrin gene.
Authors: Wiborg O , Berglund L , Boel E , Norris F , Norris K , Rehfeld J F , Marcker K A , Vuust J
Published: May 2006

A gastrin gene was isolated from a genomic library of human DNA. The human gastrin gene is about 4100 base pairs long and contains two intervening sequences. Thus, a 3500-base-pair intervening sequence is located 5 base pairs proximal to the ATG initiator codon, while a 129-base-pair intervening sequence separates the region coding for the principal hormonal form of gastrin, the heptadecapeptide, from the region coding for the major amino-terminal portion of the gastrin precursor. The 5' flanking region of the gene contains the conserved sequences, T-A-T-A-A and G-A-C-T-C-A-T-A-T, in positions similar to those of other eukaryotic genes.
The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo.
Authors: Wong S L , Price C W , Goldfarb D S , Doi R H
Published: May 2006

A cloned Bacillus subtilis gene (sprE) expressed only during the stationary growth phase is shown to encode the subtilisin E protease, an enzyme associated with sporulation. We have determined the DNA sequence of the sprE promoter region and the promoter-proximal half of the structural gene. The sprE gene codes for a putative 29-residue signal peptide and a 77-residue leader peptide preceding the mature subtilisin sequence. By plasmid integration and phage PBS1 transduction, we have mapped the sprE locus between glyB and metD on the B. subtilis chromosome, a region also containing the hyperprotease-producing hpr gene. In vitro the sprE gene is transcribed by the minor form of RNA polymerase containing a 37,000-dalton sigma factor (sigma 37). We show by S1 nuclease mapping that sprE transcription initiates at dual start sites both in vitro and in vivo and that the promoter for the downstream site has a characteristic sigma 37 recognition sequence. We propose that the physiological role of the sigma 37 RNA polymerase is to transcribe a class of genes that are catabolite repressed, that encode extracellular enzymes, or that are expressed only during the stationary phase of growth.
Adenovirus early region 1A protein activates transcription of a nonviral gene introduced into mammalian cells by infection or transfection.
Authors: Gaynor R B , Hillman D , Berk A J
Published: May 2006

Transcription from all early adenovirus promoters is stimulated by a 289 amino acid phosphoprotein encoded in the pre-early transcription unit E1A. To determine if this protein could act on a nonviral gene placed on the viral chromosome, adenovirus recombinants were constructed in which the rat preproinsulin I gene, including its promoter region, was substituted in both orientations for E1A. Preproinsulin mRNA synthesis from these recombinants was greatly stimulated after infection of line 293 cells, which constitutively express E1A protein, compared to HeLa cells, which do not. Expression of the preproinsulin gene was also greatly stimulated when HeLa cells were coinfected with the recombinants and wild-type adenovirus or a mutant defective in a second E1A protein, but much less so by coinfection with a mutant defective in the 289 amino acid phosphoprotein. Much of the E1A-induced preproinsulin mRNA had a 5' end at the same position as the preproinsulin mRNA isolated from insulinoma cells, but a considerable fraction had 5' ends mapping heterogeneously within several hundred nucleotides of this site. Preproinsulin mRNA was also detected in 293 cells but not HeLa or HEK cells after transfection of a plasmid containing the preproinsulin gene with no adenovirus sequence. This indicates that there is no cis-acting adenovirus sequence required for E1A protein stimulation of preproinsulin transcription. Infection of rat cells with adenovirus did not induce detectable mRNA synthesis from the endogenous preproinsulin I gene. These results demonstrate that the E1A protein can induce expression of a nonviral gene when it is newly introduced into mammalian cells by viral infection or transfection, but it does not induce the endogenous cellular gene.
Chaotic behavior in dopamine neurodynamics.
Authors: King R , Barchas J D , Huberman B A
Published: May 2006

We report the results of the dynamics of a model of the central dopaminergic neuronal system. In particular, for certain values of a parameter k, which monitors the efficacy of dopamine at the postsynaptic receptor, chaotic solutions of the dynamical equations appear--a prediction that correlates with the observed increased variability in behavior among schizophrenics, the rapid fluctuations in motor activity among Parkinsonian patients treated chronically with L-dopa, and the lability of mood in some patients with an affective disorder. Moreover our hypothesis offers specific results concerning the appearance or disappearance of erratic solutions as a function of k and the external input to the dopamine neuronal system.
Kindling alters the calcium/calmodulin-dependent phosphorylation of synaptic plasma membrane proteins in rat hippocampus.
Authors: Wasterlain C G , Farber D B
Published: May 2006

Septal kindling was associated with an inhibition of the post hoc phosphorylation of several synaptic plasma membrane proteins of rat hippocampus. In control rats, the 32P incorporation into proteins of molecular weights 50,000, 58,000, and 60,000 was markedly stimulated by combined calcium/calmodulin, whereas in kindled animals, the response to combined calcium/calmodulin was reduced. Calcium alone, cAMP, or cGMP modulated 32P incorporation into several synaptic plasma membrane proteins but did not differentiate control from kindled tissues. Both control and kindled rats showed nonspecific inhibition of calcium/calmodulin-stimulated phosphorylation in the post hoc assay by corticotropin and by [Leu]enkephalin. The differences between control and kindled animals were most striking in hippocampus and in the amygdaloid-entorhinal area; less pronounced in cortex, basal ganglia, and brain stem; and not significant in cerebellum, a region where kindling cannot be elicited. An 8-wk period of rest after kindling did not reduce these changes, suggesting that they may be a persistent as the kindling behavior itself.
Mercury selection of allozymes in marine organisms: Prediction and verification in nature
Authors: Nevo Eviatar , Ben-Shlomo Rachel , Lavie Batia
Published: May 2006

The geographic distributions of mercury-tolerant allozyme genotypes of the enzyme phosphoglucomutase in the shrimp
Palaemon elegans
and the enzyme phosphoglucose isomerase in the marine gastropod
Monodonta turbinata
were compared in a mercury-polluted site versus several unpolluted sites on the Israeli coast of the Mediterranean sea. We conclude that in both phosphoglucomutase and phosphoglucose isomerase, the level of the mercury-tolerant allozyme genotypes was higher in the polluted as compared with the unpolluted sites. These results suggest that mercury selection is operating in nature on allozyme genotypes of these marine organisms along patterns comparable with those found previously in laboratory experiments. We suggest that the enzymes studied here display an adaptive pattern in polluted environments. Therefore, they may be used as potential indicators and monitors of marine pollution.
Site-specific cleavage of DNA at 8- and 10-base-pair sequences.
Authors: McClelland M , Kessler L G , Bittner M
Published: May 2006

A method is described for cutting DNA at specific sites that are 8 and 10 base pairs long. The DNA is first treated with a specific methylase, either the restriction-modification enzyme M. Taq I, which converts the 4-base sequence T-C-G-A to T-C-G-mA, or the similar enzyme M. Cla I, which converts the 6-base sequence A-T-C-G-A-T to A-T-C-G-mA-T. The DNA is then cleaved with Dpn I, a restriction endonuclease that recognizes the sequence G-mA-T-C. Dpn I is unique in that it cuts only DNA that is methylated at adenine in both strands of its recognition sequence. In DNAs that are not otherwise methylated at adenine in both strands of the sequence G-A-T-C, cleavage by Dpn I occurs only at the following sequences: in the case of M. Taq I methylation, 5' T-C-G-mA - T-C-G-mA 3' 3' mA-G-C - T-mA-G-C - T 5'; in the case of M. Cla I methylation, 5' A - T-C-G-mA - T-C-G-mA-T 3' 3' T-mA-G-C - T-mA-G-C - T-A 5'. Specific cutting and cloning at these methylase/Dpn I-generated sites is shown experimentally. Further, we describe how the above technique can be extended to generate Dpn I cleavage sites of up to 12 base pairs. In DNA that contains equal amounts of each base distributed at random, 8- and 10-base-pair recognition sequences occur, on the average, approximately once every 65,000 and 1,000,000 base pairs, respectively. Potential applications, including the development of cloning vectors and a rapid method for chromosome walking, are discussed.
Molecular cloning of a unique human T-cell leukemia virus (HTLV-IIMo).
Authors: Gelmann E P , Franchini G , Manzari V , Wong-Staal F , Gallo R C
Published: May 2006

Human T-cell leukemia virus IIMo (HTLV-IIMo) is a human retrovirus isolated from a patient with a T-cell hairy cell leukemia. This virus has been shown to have core protein (gag) antigens similar to, but distinct from, those of all known isolates of the prototype human T-cell leukemia virus (HTLV-I). We have used a subgenomic clone of the HTLV-I env-pX region to detect and characterize HTLV-IIMo proviral sequences by performing Southern blot hybridization under conditions of low stringency. Using the HTLV-I probe, we cloned a partial integrated HTLV-IIMo provirus from a genomic library of the producer Mo cell line. These sequences could be characterized by low stringency hybridization with different subgenomic clones of HTLV-I. An HTLV-IIMo-specific subclone was made by isolating a 3.6-kilobase BamHI fragment of the partial provirus. This was used to clone two full-length integrated viral genomes. Using the HTLV-IIMo viral probe, we also showed by hybridization under stringent conditions to DNA and RNA of various infected and uninfected cell lines that these HTLV-IIMo sequences are unique.
Monitoring of cytosolic free Ca2+ in C5a-stimulated neutrophils: loss of receptor-modulated Ca2+ stores and Ca2+ uptake in granule-free cytoplasts.
Authors: Gennaro R , Pozzan T , Romeo D
Published: May 2006

The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intracellular Ca2+ indicator quin2, 2-[[2-bis(acetylamino)-5-methylphenoxy]methyl-6-methoxy-8- bis(acetylamino)]quinoline. Neutrophils at rest have a cytosolic Ca2+ concentration of 85 +/- 5 nM, which in 2-4 min increases to 300-400 nM upon interaction with the complement fragment C5a in a concentration range of 35 pM to 1.2 microM. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 less than 0.5 nM), O-2 production (ED50 = 9 nM), and secretion of the contents of specific granules (ED50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration above 1 microM under conditions where it stimulates neutrophil functions. Conversely, phorbol 12-myristate 13-acetate markedly activates secretion and O-2 production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA (Ca2+out approximately equal to 20 nM), with both C5a and ionomycin, cytosolic Ca2+ increases to less than 200 nM, and functional responses are greatly decreased. Nucleus- and granule-free neutrophil cytoplasts accumulate Ca2+ and produce O-2 when exposed to ionomycin but not to C5a. These results and other considerations suggest that (i) activation of neutrophil functions may occur after cytosolic Ca2+ has exceeded the apparent threshold level of 200 nM; (ii) C5a receptor-mediated activation of Ca2+ influx may require cooperation between the neutrophil surface and some cytoplasmic organelle and/or redistribution of the C5a-receptor complexes on the cell surface; and (iii) the phorbol diester stimulates Ca2+-dependent pathways presumably by directly activating other mechanisms such as protein phosphorylation.
Triggering of the T3-Ti antigen-receptor complex results in clonal T-cell proliferation through an interleukin 2-dependent autocrine pathway.
Authors: Meuer S C , Hussey R E , Cantrell D A , Hodgdon J C , Schlossman S F , Smith K A , Reinherz E L
Published: May 2006

Human T-cell clones and anti-T-cell-receptor antibodies (clonotypic) directed at surface receptors for antigen (T3-Ti molecular complex) as well as anti-interleukin 2 (IL-2) and anti-IL-2-receptor antibodies were utilized to investigate the mechanism by which alloantigens or antigen plus self-major histocompatibility complex (MHC) (i.e., physiologic ligand) trigger specific clonal proliferation. Soluble or Sepharose-bound anti-Ti monoclonal antibodies, like physiologic ligand, enhanced proliferative responses to purified IL-2 by inducing a 6-fold increase in surface IL-2 receptor expression. In contrast, only Sepharose-bound anti-Ti or physiologic ligand triggered endogenous clonal IL-2 production and resulted in subsequent proliferation. The latter was blocked by antibodies directed at either the IL-2 receptor or IL-2 itself. These results suggest that induction of IL-2 receptor expression but not IL-2 release occurs in the absence of T3-Ti receptor cross-linking. Perhaps more importantly, the findings demonstrate that antigen-induced proliferation is mediated through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors.
The pre-melt phase of
n
-alkanes: Crystallographic evidence for a kinked chain structure
Authors: Dorset D. L. , Moss B. , Wittmann J. C. , Lotz B.
Published: May 2006

Electron diffraction measurements on epitaxially grown crystals of orthorhombic
n
-hexatriacontane,
n
-C
36
H
74
, give evidence for the chain-defect mechanism for linear chain melting. The derived structural model is also in accord with recent spectroscopic studies of odd-chain
n
-alkanes, and the diffraction data specifically exclude models based on helices or rigid chain rotors. Stability of a kinked chain structure, moreover, is indicated by an observed hysteresis effect that gives different pretransition temperatures for solution-grown and annealed crystals.
Isolation of a collagenase cDNA clone and measurement of changing collagenase mRNA levels during induction in rabbit synovial fibroblasts.
Authors: Gross R H , Sheldon L A , Fletcher C F , Brinckerhoff C E
Published: May 2006

To facilitate our studies on the mechanisms controlling collagenase production at a molecular level in rabbit synovial fibroblasts, we have constructed a cDNA library using mRNAs isolated from cells induced with crystals of monosodium urate monohydrate. We have screened this library with cDNA probes made from induced and control mRNA populations. From among 30 clones that hybridized preferentially to the induced-cell probe, 4 contained collagenase sequences. The largest, a clone of 650 base pairs, was identified by its ability to hybrid select a mRNA that could be translated in a cell-free system into a product that was precipitable with monospecific antibody to collagenase. Using this clone to probe blots of RNA from induced cells, we detected the appearance of a collagenase mRNA of 2.7 kilobases within 5 hr of addition of urate. The level of collagenase mRNA continued to increase for 35-40 hr, when it was 60 to 90 times more abundant in induced cells than in control cells. The increase in mRNA levels correlated with an increase in immunoreactive collagenase protein that was detectable in culture medium by 10 hr.
Cloning and expression of the gene for bacteriophage T7 RNA polymerase.
Authors: Davanloo P , Rosenberg A H , Dunn J J , Studier F W
Published: May 2006

The complete coding sequence of the gene for bacteriophage T7 RNA polymerase (T7 gene 1) has been cloned in the plasmid pBR322. Large amounts of active enzyme can be accumulated in Escherichia coli when the cloned gene is transcribed from the lac UV5 promoter. A protease activity that apparently can nick the protein without causing it to fall apart can be a problem during purification, but a procedure is described that gives good yields of essentially homogeneous, highly active enzyme suitable for biochemical and physical studies. T7 RNA polymerase has a stringent specificity for its own promoters and will selectively transcribe DNA that has been linked to such a promoter. This specificity makes the enzyme useful both for producing specific RNAs in vitro and for directing the expression of selected genes inside the cell. Having the cloned gene also makes possible a detailed mutational analysis of the functioning of T7 RNA polymerase.
Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen.
Authors: Brady J , Bolen J B , Radonovich M , Salzman N , Khoury G
Published: May 2006

The early simian virus 40 (SV40) gene product, large tumor (T) antigen, is responsible for the initiation of viral DNA replication and the autoregulation of early gene expression through direct protein-DNA interactions. We investigated the role of T antigen in late viral gene expression, independent of its function in amplifying templates through DNA replication. SV40 DNA was transfected into BSC-1 and COS-1 cells and cultured in the presence of inhibitors of DNA replication. Electrophoretic immunoblot analysis indicated that both the onset and the extent of SV40 late gene expression is increased in COS-1 cells, which constitutively express SV40 T antigen. Blot hybridization analysis of poly(A)-selected RNA demonstrated that the level of synthesis of the major late structural protein VP-1 in COS-1 cells was due to increased transcription. Similar results were obtained when plasmids that contain the SV40 late gene but lack both the origin for viral DNA replication and the early gene coding region were transfected onto COS-1 cells. Using lines of SV40-transformed monkey kidney cells that express altered T antigens, we found that enhanced expression of the late gene product is correlated with the ability of T antigen to bind SV40 DNA. These results indicate that large T antigen plays a role in the stimulation of late viral gene expression.
Continuous suppression of globin gene expression and differentiation of Friend erythroleukemia cells by phorbol 12-myristate 13-acetate (PMA) despite the loss of PMA binding sites by down regulation.
Authors: Yamasaki H , Martel N , Fusco A , Ostertag W
Published: May 2006

The tumor promoter phorbol 12-myristate 13-acetate (PMA) reversibly inhibits hexamethylene bisacetamide-induced terminal differentiation of Friend erythroleukemia cells (FELC). We were successful in continuously inhibiting FELC differentiation by PMA up to 125 weeks (about 240 serial passages of cells in the presence of PMA). During that period, FELC can be induced to differentiate and enter terminal cell division upon removal of PMA. PMA-mediated suppression of FELC differentiation was associated with only a low level of globin mRNA accumulation. However, a rapid accumulation of globin mRNA in the cytoplasm followed by hemoglobin accumulation occurred upon removal of PMA. A specific, saturable, high-affinity receptor for phorbol esters is present in FELC, as was shown by binding studies with [3H]phorbol 12,13-dibutyrate. A significant (80%) loss in the number of phorbol ester receptors of FELC was observed after a continuous inhibition of differentiation by PMA for as much as 125 weeks. Despite such a down regulation of phorbol ester receptors, these cells respond to PMA with a dose-response similar to that of their parent cells, which have the normal number of phorbol ester receptors. Thus, PMA can suppress reversibly the accumulation of globin-specific mRNA and terminal differentiation of FELC during prolonged periods, despite loss of receptor sites, and our results suggest that only few phorbol ester receptors may be necessary for complete inhibition of FELC differentiation by PMA.
Molecular genetics of a biological clock in
Drosophila
Authors: Bargiello Thaddeus A. , Young Michael W.
Published: May 2006

Ninety kilobase pairs (kb) of DNA have been isolated from the 3B region of the X chromosome of
Drosophila melanogaster
. Previous cytogenetic analyses have placed a gene required for rhythmic behavior (
per
) in this chromosomal interval. Physical characterization of a series of chromosomal rearrangements altering
per
locus activity indicates that DNA affecting behavioral rhythms is found in a 7.1-kb
Hin
dIII fragment. A single 4.5-kb poly(A)
+
RNA is transcribed from this DNA in wild-type pupae and adult flies. The transcript is eliminated by a
per
mutant that retains some rhythmic activity, but this mutant substitutes two novel transcripts, 11.5 kb and 0.9 kb. It is suggested that the new poly(A)
+
transcripts provide residual
per
locus activity.
Somatic diversification of immunoglobulins.
Authors: Rudikoff S , Pawlita M , Pumphrey J , Heller M
Published: May 2006

A series of three IgM, kappa monoclonal antibodies arising from a fusion of BALB/c spleen cells from mice immunized with beta-(1,6)-galactan-containing antigens have been analyzed. These three lines were found (i) to have homologous protein sequences in the heavy chain D region and at the sites of recombination between the heavy chain variable and D segment (VH-D) and the D and joining segment (D-JH), although amino acid substitutions were observed in both the heavy and light chain variable regions; (ii) to use identical heavy and light chain joining segments; and (iii) to demonstrate two identical (productive and nonproductive) kappa-chain rearrangements. A likely explanation for these observations is that the three lines are clonally related (arise from a common precursor) and that the observed heavy and light chain variable segment substitutions represent somatic point mutations. Because these antibodies are all of the IgM class, the results indicate that a somatic mutational mechanism is activated early in B-cell ontogeny and operates at both the heavy and light chain loci. Furthermore, the somatic mutation process appears to continue during the development of a given cell line, but is independent of class switching.
Epitope-specific protective immunogenicity of chemically synthesized 13-, 18-, and 23-residue peptide fragments of streptococcal M protein.
Authors: Beachey E H , Tartar A , Seyer J M , Chedid L
Published: May 2006

The ability of chemically synthesized subpeptides of type 24 streptococcal M protein to evoke protective antibodies in rabbits was investigated. We synthesized copies of the COOH-terminal 13, 18, and 23 amino acid residues of cyanogen bromide fragment 7 (CB7) of pepsin-extracted type 24 M protein, except that methionine was substituted for homoserine as the COOH-terminal residue. An additional residue of cysteine was added at the COOH terminus of the 13-residue peptide. Each of the peptides, designated S-CB7-(23-35)-Cys, S-CB7-(18-35), and S-CB7-(13-35), when conjugated to lysylated tetanus toxoid with glutaraldehyde, was capable of stimulating formation of protective anti-type 24 M protein antibodies in rabbits. The smallest peptide, S-CB7-(23-35)-Cys, elicited immune responses equally as strong, if not stronger, than those to the longer peptides. A single Lys/Gly substitution in this 13-residue peptide resulted in its failure to stimulate protective antibodies. None of the antisera reacted with heterologous serotypes of M protein and none reacted with frozen sections of human heart tissue. These results indicate that a chemically synthesized peptide fragment corresponding to as few as 13 amino acid residues of streptococcal M protein is capable of evoking protective anti-streptococcal antibodies without evoking antibodies crossreactive with cardiac tissue.
Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.
Authors: Peerce B E , Wright E M
Published: May 2006

Fluorescein isothiocyanate (FITC) was used to label the rabbit intestinal brush border Na+-glucose carrier, identify the carrier protein on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and monitor the effect of ions and substrates on fluorescence quenching. Enriched brush border preparations were employed to study both glucose transport and FITC binding. FITC and a nonfluorescent analog (phenyl isothiocyanate, PITC) both inhibited Na+-dependent D-glucose transport irreversibly. Inhibition was blocked completely by the presence of Na+ and D-glucose during labeling. PITC was used to label nonspecific amino groups in the presence of glucose and Na+, and then the glucose carrier was labeled with FITC in the absence of substrates. Fluorescence of FITC bound to the carrier was quenched specifically with Na+ in a saturable fashion, and this indicates a Na+-dependent conformational change in the carrier. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of FITC-labeled membranes revealed specific labeling of a 71,000-dalton peptide. We conclude that Na+ induces a conformational shift in the 71,000-dalton glucose carrier, and this is quite consistent with the kinetics of Na+-dependent glucose transport in these membranes.
High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector.
Authors: Jay E , Rommens J , Pomeroy-Cloney L , MacKnight D , Lutze-Wallace C , Wishart P , Harrison D , Lui W Y , Asundi V , Dawood M
Published: May 2006

A chemically synthesized gene for human interferon-gamma has been cloned into a prokaryotic expression vector under the regulation of a synthetic constitutive transcriptional-translational control unit that contains a strong bacteriophage T5 early promoter and a strong ribosome-binding site. Cells harboring the recombinant plasmid express high levels (4 X 10(9) units per liter of culture) of antiviral activity specific for interferon-gamma. Analysis of total cell lysates on NaDodSO4/polyacrylamide gels revealed a 17,200-dalton protein, expected for the nonglycosylated form of human interferon-gamma, that constitutes greater than 15% of total cell protein.
Immunological evidence for the in vivo occurrence of (2'-5')adenylyladenosine oligonucleotides in eukaryotes and prokaryotes.
Authors: Laurence L , Marti J , Roux D , Cailla H
Published: May 2006

A monoclonal antibody highly specific for (2'-5')adenylyladenosine oligonucleotides was used together with a 125I-labeled analog of this compound to detect and quantify phosphorylated and nonphosphorylated (2'-5')adenylyladenosine oligonucleotides in a variety of tissues and cells. These oligonucleotides were first assayed as a whole in perchloric acid extracts and then further individually characterized by HPLC analysis. Their sensitivity to alkaline phosphatase, snake venom phosphodiesterase, and T2 RNase was systematically checked. Nonphosphorylated (2'-5')adenylyladenosine oligonucleotides were found in mammalian tissues as well as in yeast and bacteria. In normal mouse brain, lung, heart, pancreas, spleen, kidney, and liver their concentrations ranged from 10 to 200 pmol/g wet weight, depending on tissue and strain. The oligonucleotides were mainly dimers, trimers, tetramers, and pentamers. In addition, phosphorylated (2'-5')adenylyladenosine oligonucleotides were shown in liver and kidney extracts.