bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and sequence of a human cytochrome P-450 cDNA clone.
Authors: Phillips I R , Shephard E A , Ashworth A , Rabin B R
Published: May 2006

A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family.
Identification of an active site peptide of skeletal myosin after photoaffinity labeling with N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate.
Authors: Okamoto Y , Yount R G
Published: May 2006

The active site of skeletal myosin has been photoaffinity labeled (approximately equal to 50%) by the ADP analog N-(4-azido-2-nitrophenyl)-2-aminoethyl triphosphate (NANDP) following the cobalt phenanthroline active site trapping procedure of Wells and Yount [Wells, J. A. & Yount, R. G. (1979) Proc. Natl. Acad. Sci. USA 76, 4966-4970]. Extensive proteolytic digestion of [3H]NANDP-labeled myosin subfragment one yielded two major peptides, P1 and P2, which were purified by reversed-phase high-performance liquid chromatography. These peptides represented 50% of all labeled amino acids and contained 1 mol of the unusual amino acid epsilon-N-trimethyllysine. Analysis of P2 by Edman techniques gave a sequence Val-Asn-Pro-Tyr-Lys(Me3)-X-Leu-Pro-Val-Tyr, which corresponds to an identical sequence for residues 125-134 determined by Tong and Elzinga [Tong, S. W. & Elzinga, M. (1983) J. Biol. Chem. 258, 13100-13110] for a segment of rabbit skeletal myosin heavy chain in which X is Trp-130. P1 was identical to P2 except it contained an additional three amino acids, Asn-Pro-Gln, at the COOH-terminal end. Amino acid composition, sequence data, spectral measurements, and location of radioactive label in both P1 and P2 all indicate Trp-130 is the major site of labeling by NANDP. The adjacent epsilon-N-trimethyllysine may provide part of the binding site for the triphosphate portion of ATP.
The NADPH binding site on beef liver catalase.
Authors: Fita I , Rossmann M G
Published: May 2006

Beef liver and human erythrocyte catalases (EC 1.11.1.6) bind NADP tenaciously [Kirkman, H. N. & Gaetani, G. F. (1984) Proc. Natl. Acad. Sci. USA 81, 4343-4348]. The position of NADP on beef liver catalase corresponds to the carboxyl-terminal polypeptide hinge in Penicillium vitale fungal catalase, which connects the common catalase structure to the additional flavodoxin-like domain. In contrast to nearly all other known structures of protein-bound NADP, NAD, and FAD, the NADP molecule of beef liver catalase is folded into a right-handed helix and bound, in part, in the vicinity of the carboxyl end of two alpha-helices. A water molecule (W7) occupies a pseudosubstrate site close to the C4 position of the nicotinamide and is hydrogen bonded to His-304. Although the NADP and heme groups approach each other to within 13.7 A, there is no direct interaction. The function of the NADP remains a mystery.
Genes specifying degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4.
Authors: Ghosal D , You I S , Chatterjee D K , Chakrabarty A M
Published: May 2006

All of the structural genes for 3-chlorobenzoate degradation are clustered in a 4.2-kilobase (kb) region of plasmid pAC25 (or pAC27) in Pseudomonas putida. An approximate 10-kb DNA segment containing three structural genes for chlorocatechol metabolism present on plasmid pJP4 in Alcaligenes eutrophus shows homology with the above 4.2-kb region of pAC27. In spite of the detectable sequence homology in the structural genes present on both plasmids, the regulation of their expression seems quite different; unlike pAC27, structural rearrangements are prerequisite for efficient expression of the 3-chlorobenzoate genes on plasmid pJP4. Structural features such as stem-loop structures present on plasmid pJP4 are most likely the starting materials for such rearrangements.
Structure at 2.9-A resolution of aspartate carbamoyltransferase complexed with the bisubstrate analogue N-(phosphonacetyl)-L-aspartate.
Authors: Krause K L , Volz K W , Lipscomb W N
Published: May 2006

In an x-ray diffraction study by the isomorphous replacement method, the structure of the complex of aspartate carbamoyltransferase (EC 2.1.3.2) bound to the bisubstrate analogue N-(phosphonacetyl)-L-aspartate has been solved to 2.9-A resolution (R = 0.24). The large quaternary structural changes previously deduced by molecular replacement methods have been confirmed: the two catalytic trimers (c3) move apart by 12 A and mutually reorient by 10 degrees, and the regulatory dimers (r2) reorient each about its twofold axis by about 15 degrees. In this, the T-to-R transition, new polar interactions develop between equatorial domains of c chains and the Zn domain of r chains. Within the c chain the two domains, one binding the phosphonate moiety (polar) and the other binding the aspartate moiety (equatorial) of the inhibitor N-(phosphonacetyl)-L-aspartate, move closer together. The Lys-84 loop makes a large relocation so that this residue and Ser-80 bind to the inhibitor of an adjacent catalytic chain within c3. A very large change in tertiary structure brings the 230-245 loop nearer the active site, allowing Arg-229 and Gln-231 to bind to the inhibitor. His-134 is close to the carbonyl group of the inhibitor, and Ser-52 is adjacent to its phosphonate group. However, no evidence exists in the literature for phosphorylation of serine in the mechanism. Residues studied by other methods, including Cys-47, Tyr-165, Lys-232, and Tyr-240, are too far from the inhibitor to have a direct interaction.
Rapid purification of cytosolic epoxide hydrolase from normal and clofibrate-treated animals by affinity chromatography.
Authors: Prestwich G D , Hammock B D
Published: May 2006

Epoxide hydrolase from liver cytosol (cEH) of both normal and clofibrate-treated mice can be bioselectively adsorbed onto an affinity column prepared from epoxy-activated Sepharose and 7-methoxycitronellyl thiol. The free ligand is a modest inhibitor of cEH (I50, approximately equal to 3 X 10(-4) M) and lacks the epoxide function necessary for it to be turned over as a substrate. This study demonstrates that a methoxy group can be used to mimic an oxirane in a vertebrate system. Bioselective elution of cEH can be accomplished with several chalcone oxides, which are selective potent inhibitors (I50, 1-50 X 10(-7) M), and activity can be recovered by dialysis. This procedure thus enhances the purification by offering independent opportunities for selective binding and selective elution. Conservatively, a 40%-80% recovery of partially inhibited enzyme activity can be achieved in 4-48 hr with a 30- to 90-fold purification. The purified cEH from clofibrate-induced animals was essentially homogeneous by NaDodSO4/PAGE and had an apparent subunit molecular weight of 58,000. The cEHs from normal and clofibrate-induced animals appeared identical by NaDodSO4/PAGE. Since the cEH activity in normal and clofibrate-treated animals is due to the same enzyme, the increase in cEH activity caused by selected hypolipidemic agents appears to be true induction.
An arylaminopyridazine derivative of gamma-aminobutyric acid (GABA) is a selective and competitive antagonist at the GABAA receptor site.
Authors: Chambon J P , Feltz P , Heaulme M , Restle S , Schlichter R , Biziere K , Wermuth C G
Published: May 2006

In view of finding a new gamma-aminobutyric acid (GABA) receptor ligand we synthesized an arylaminopyridazine derivative of GABA, SR 95103 [2-(carboxy-3'-propyl)-3-amino-4-methyl-6-phenylpyridazinium chloride]. SR 95103 displaced [3H]GABA from rat brain membranes with an apparent Ki of 2.2 microM and a Hill number near 1.0. SR 95103 (1-100 microM) antagonized the GABA-mediated enhancement of [3H]diazepam binding in a concentration-dependent manner without affecting [3H]diazepam binding per se. SR 95103 competitively antagonized GABA-induced membrane depolarization in rat spinal ganglia. In all these experiments, the potency of SR 95103 was close to that of bicuculline. SR 95103 (100 microM) did not interact with a variety of central receptors--in particular the GABAB, the strychnine, and the glutamate receptors--did not inhibit Na+-dependent synaptosomal GABA uptake, and did not affect GABA-transaminase and glutamic acid decarboxylase activities. Intraperitoneally administered SR 95103 elicited clonicotonic seizures in mice (ED50 = 180 mg/kg). On the basis of these results it is postulated that St 95103 is a competitive antagonist of GABA at the GABAA receptor site. In addition to being an interesting lead structure for the search of GABA ligands, SR 95103 could also be a useful tool to investigate GABA receptor subtypes because it is freely soluble in water and chemically stable.
From monoclonal antibody to gene for a neuron-specific glycoprotein in Drosophila.
Authors: Zipursky S L , Venkatesh T R , Benzer S
Published: May 2006

A monoclonal antibody (MAb24B10), derived from mice immunized with Drosophila retina, exclusively stains photoreceptor cells in the retina and their axonal projections to the optic ganglia. The antigen (Ag24B10) is a 160-kDa glycoprotein comprising about 0.8% of the retina protein. By microsequencing, 19 of the first 21 amino acids at the NH2-terminal end of the protein have been determined. Using synthetic oligonucleotide probes corresponding to a portion of this amino acid sequence, we isolated a homologous lambda genomic clone. A partial DNA sequence of this clone, along with blot experiments on genomic DNA and RNA, indicate that this clone is part of the structural gene for Ag24B10. By in situ hybridization, the gene was localized to the tip of chromosome 3R.
Autoregulation of collagenase production by a protein synthesized and secreted by synovial fibroblasts: cellular mechanism for control of collagen degradation.
Authors: Brinckerhoff C E , Benoit M C , Culp W J
Published: May 2006

Conditioned medium taken from cultures of resting rabbit synovial fibroblasts contained a protein that prevented the synthesis of the neutral proteinase collagenase. Conditioned medium was concentrated 10-fold and placed on cultures of rabbit synovial fibroblasts along with an inducer of collagenase (phorbol myristate acetate or latex particles) and [3H]leucine. Collagenase production was measured by immunoprecipitation of culture medium with monospecific antibody. Gel filtration showed that the inhibitory factor had MrS of 12,500, 25,000-50,000, and 150,000, suggesting that the protein may exist as aggregates. Activity was destroyed by boiling, by trypsin, and by dithiothreitol. Production of the inhibitory protein was prevented by cycloheximide. Isoelectric focusing purified the protein 100- to 150-fold and revealed pIs in the range of 3.2-3.7. Glycosylation was demonstrated by binding to Con A-Sepharose. Our data indicate that rabbit synovial fibroblasts autoregulate collagenase production and suggest that the low levels of collagenase seen in resting cultures result from an active suppression of collagenase synthesis.
Sequence analysis of hepatitis A virus cDNA coding for capsid proteins and RNA polymerase.
Authors: Baroudy B M , Ticehurst J R , Miele T A , Maizel J V , Purcell R H , Feinstone S M
Published: May 2006

We report here the nucleotide sequence corresponding to two large regions of the hepatitis A virus (HAV) genome. These comprise a sequence of 3274 bases corresponding to the 5' end of the genome, which includes the putative capsid protein region of this picornavirus, and 1590 bases corresponding to the 3' end of the genome, terminating in a 15-base poly(A) tract. These sequences revealed that HAV had the characteristic genomic organization of picornaviruses: an open reading frame beginning approximately 750 bases from the 5' end of the RNA and a termination codon 60 bases from the 3' poly(A) tract. The predicted amino acid sequences of both regions have been compared to analogous regions previously determined for other picornaviruses. There was sufficient homology to conclude that the 5' region of HAV codes for capsid proteins and that the 3' region codes for an RNA polymerase. However, these regions of HAV were not found to be closely related to analogous regions of poliovirus, encephalomyocarditis virus, and foot and mouth disease virus.
Cloning and sequencing of cDNA encoding the complete mouse liver alcohol dehydrogenase.
Authors: Edenberg H J , Zhang K , Fong K , Bosron W F , Li T K
Published: May 2006

The main ethanol-active alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) in mouse liver (ADH-AA) is similar in catalytic and molecular properties to horse liver ADH-EE and to the human class I ADHs. We have isolated cDNA clones encoding the entire mouse liver enzyme plus flanking regions. A mixture of 16 different oligonucleotides, each 14 bases long, was used to screen a liver cDNA library made from a DBA/2J mouse. A strongly hybridizing clone was found and identified as an ADH-encoding cDNA by partial DNA sequencing. This clone was used as a probe to identify others. Two overlapping cDNA clones together contained the entire protein-encoding region plus 100 nucleotides of the 5' noncoding region and 133 nucleotides of the 3' noncoding region culminating in a short poly(dA) tail. The amino acid sequence of the mouse liver enzyme deduced from this cDNA closely resembles that of horse liver ADH-E: 316 of 374 residues are identical, and 29 of the differences are conservative substitutions. The 5' region of this cDNA is interesting: the AUG that initiates the ADH polypeptide is preceded by an AUG that would encode the first amino acid of a tripeptide. Presumably termination of this tripeptide is followed by reinitiation at the AUG immediately preceding the sequence of the mature ADH polypeptide.
Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.
Authors: Kan C C , Solomon E , Belt K T , Chain A C , Hiorns L R , Fey G
Published: May 2006

Six alpha 2-macroglobulin (alpha 2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, p alpha 2M1, carries a 4.6 kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature alpha 2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor pro-alpha 2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published alpha 2M amino acid sequence for all except three residues. The alpha 2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using alpha 2M cDNA as a hybridization probe.
Deletions within a hinge region of a specific DNA-binding protein.
Authors: Little J W , Hill S A
Published: May 2006

Many proteins are organized as a set of compact functional domains connected by flexible, exposed segments of the polypeptide chain. To study one of these connector regions, we isolated a series of functional in-frame deletions in the central portion of a specific DNA-binding protein, the LexA repressor of Escherichia coli. These mutant proteins fell into two main classes: those with small deletions of two to eight amino acids functioned as repressor about as well as did wild type, while those with large deletions of 17-22 amino acids functioned well only at considerably higher concentrations. The mutant proteins were resistant to the specific cleavage reaction that triggers the SOS response. These data suggest that the conformation of the hinge region in LexA protein is important for cleavage. By contrast, the hinge plays a topological role in repressor function, connecting the two functional halves of the protein; in the SOS response, this function of the hinge is inactivated by cleavage, leading to inactivation of the repressor.
Phosphorylation of the calcium antagonist receptor of the voltage-sensitive calcium channel by cAMP-dependent protein kinase.
Authors: Curtis B M , Catterall W A
Published: May 2006

Physiological studies indicate that voltage-sensitive calcium channels are regulated by cAMP and protein phosphorylation. The calcium antagonist receptor of the voltage-sensitive calcium channel from transverse-tubule membranes consists of three subunits, designated alpha, beta, and gamma. The catalytic subunit of cAMP-dependent protein kinase phosphorylates both the alpha and beta subunits of the purified receptor at a rate and extent that suggests they are potential physiological substrates of this enzyme. The phosphorylation of the alpha and beta subunits in transverse-tubule membranes was analyzed by two-dimensional gel electrophoresis. In intact transverse-tubule membranes, the alpha subunit is not significantly phosphorylated. However, the beta subunit, identified by its Mr, pI, and binding to wheat germ agglutinin-Sepharose, was one of the substrates selectively phosphorylated by cAMP-dependent protein kinase in transverse-tubule membranes. These results suggest that cAMP-dependent phosphorylation of the beta subunit of the calcium antagonist receptor may be an important regulatory mechanism for calcium channel function.
Characterization of [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin binding to delta opiate receptors in the rat brain and neuroblastoma--glioma hybrid cell line (NG 108-15).
Authors: Akiyama K , Gee K W , Mosberg H I , Hruby V J , Yamamura H I
Published: May 2006

Specific binding properties of the tritium-labeled delta opiate receptor agonist [3H][2-D-penicillamine, 5-D-penicillamine]enkephalin [( 3H][D-Pen2, D-Pen5]enkephalin) were characterized in the rat brain and in a mouse neuroblastoma-rat glioma hybrid cell line (NG 108-15). Saturation isotherms of [3H][D-Pen2, D-Pen5]enkephalin binding to rat brain and NG 108-15 membranes gave apparent Kd values of 1-6 nM. These values are in good agreement with the Kd value obtained from the kinetic studies. The Bmax value in NG 108-15 membranes was 235.3 fmol/mg of protein. An apparent regional distribution of [3H][D-Pen2, D-Pen5]enkephalin binding was observed in the rat brain. A number of enkephalin analogues inhibited [3H][D-Pen2, D-Pen5]enkephalin binding with high affinity (IC50 values of 0.5-5.0 nM) in both NG 108-15 and rat brain membranes. However, putative mu receptor-selective ligands such as morphine, [D-Ala2, MePhe4, Gly5-ol]enkephalin, [MePhe3, D-Pro4]morphiceptin, and naloxone were less effective inhibitors of [3H][D-Pen2, D-Pen5]enkephalin binding in both systems tested. These data suggest that [3H][D-Pen2, D-Pen5]enkephalin is a potent and selective ligand for the delta opioid receptor.
Associative conditioning analog selectively increases cAMP levels of tail sensory neurons in Aplysia.
Authors: Ocorr K A , Walters E T , Byrne J H
Published: May 2006

Bilateral clusters of sensory neurons in the pleural ganglia of Aplysia contain cells involved in a defensive tail withdrawal reflex. These cells exhibit heterosynaptic facilitation in response to noxious skin stimulation that can be mimicked by the application of serotonin. Recently it has been shown that this facilitation can be selectively amplified by the application of a classical conditioning procedure to individual sensory neurons. We now report that an analog of this classical conditioning paradigm produces a selective amplification of the cAMP content of isolated sensory neuron clusters. The enhancement is achieved within a single trial and appears to be localized to the sensory neurons. These results indicate that a pairing-specific enhancement of cAMP levels may be a biochemical mechanism for associative neuronal modifications and perhaps learning.
1,2-Diacylglycerol and phorbol ester inhibit agonist-induced formation of inositol phosphates in human platelets: possible implications for negative feedback regulation of inositol phospholipid hydrolysis.
Authors: Watson S P , Lapetina E G
Published: May 2006

The present study has demonstrated that pretreatment of human platelets with either phorbol ester or 1,2-diacylglycerol inhibits agonist-induced formation of inositol phosphates; this inhibition can be correlated with a decrease in the release of ATP and 5-hydroxytryptamine by thrombin. The mechanism of this action is not known, but a role for protein kinase C is suggested, as both phorbol ester and 1,2-diacylglycerol have in common the ability to activate this enzyme. These results have important implications as a possible negative feedback control over agonist-induced hydrolysis of inositol phospholipids.
A monoclonal antibody disrupting calcium-dependent cell-cell adhesion of brain tissues: possible role of its target antigen in animal pattern formation.
Authors: Hatta K , Okada T S , Takeichi M
Published: May 2006

The Ca2+-dependent cell-cell adhesion system (CDS) is thought to be essential for the formation and maintenance of cell adhesion in a wide variety of tissues. Previous studies suggested that CDS has some cell-type specificity; for example, the monoclonal antibody ECCD-1 selectively recognizes CDS of certain epithelial tissues in mouse embryos but not nervous tissues. In the present study, we have obtained a monoclonal antibody, designated NCD-1, that disrupts connections between brain cells of mouse embryos. A series of experiments suggested that NCD-1 specifically recognizes CDS. We then determined the distribution of the NCD-1 antigen in various mouse tissues. NCD-1 reacted with cells of the following tissues and cell lines: nervous tissues from various sources, lens, striated muscle, cardiac muscle, glioma G26-20, adrenocortical tumor Y1, and melanoma B16. None of these cells reacted with ECCD-1, and the cells reactive with ECCD-1 did not react with NCD-1. There was also a class of cells that did not react with either ECCD-1 or NCD-1. These results suggest that cells in the body can be classified into at least three groups containing CDS of differing specificities. A map of the tissue localization of these different classes of CDS also suggests that the expression of cell-type-specific cell adhesion molecules in each tissue plays a crucial role in adhesion between the same cell types and segregation of different cell types in processes essential for animal morphogenesis.
Ontogeny of hamster hemoglobins in yolk-sac erythroid cells in vivo and in culture.
Authors: Boussios T , Condon M R , Bertles J F
Published: May 2006

During mammalian hemoglobin ontogeny, synthesis of the earliest globin chains (embryonic) is ultimately replaced by synthesis of globin chains (adult) characteristic of the fully formed organism. Elements of control of initiation, progression, and completion of globin-chain ontogeny are poorly understood. In search of a cell culture system in which ontogeny might be studied under closely controlled experimental conditions, we chose erythroid cells of the hamster embryo. First, the ontogeny of globin chains was defined in these yolk-sac-derived erythroid cells from day 10 through day 13 in gestation. Amounts of individual embryonic and adult globin chains were quantified, as were their rates of synthesis. Next, analogous studies were performed on yolk-sac erythroid cells from day 10 in gestation (prior to the appearance of fetal liver) grown in culture for 3 days, corresponding to days 10-13 in vivo. The ontogenic program in culture was virtually identical to that in vivo. Approximately 70% of active globin synthesis was embryonic at day 10 in gestation (day 0 of culture), declining to 30% by day 13 in gestation (day 3 of culture). Whereas only trace synthesis of the adult non-alpha chains (beta major and beta minor) were initially observed, their combined active synthesis achieved a level of approximately 30% 3 days later both in vivo and in culture. Cell hemoglobin content and cell morphology were similar in both systems. We conclude that an ontogenic program for globin-chain synthesis exists in these primitive erythroid cells, overriding possible influences of cell environment. Further, we suggest that these cells in culture provide a means of examining cell mechanisms associated with globin-gene ontogeny under controlled experimental conditions.
Evolution of mouse major histocompatibility complex genes borne by t chromosomes.
Authors: Figueroa F , Golubić M , Nizetić D , Klein J
Published: May 2006

Virtually all wild mouse populations carry t haplotypes that cause embryonic lethality or semilethality, distortion of segregation ratios, suppression of crossing-over, and male sterility. The t complex of genes is located on chromosome 17, closely linked to the H-2, the major histocompatibility complex of the mouse. The t haplotypes differ from each other not only in lethal genes they carry but also in their linked H-2 haplotypes. In this study, we compared the class II H-2 genes present on 31 t chromosomes extracted from wild populations in different parts of the world. The comparison was based on the analysis of DNA fragments obtained after digestion with restriction endonucleases. The results reveal the existence of three major groups of class II alleles representing main branches on the evolutionary tree of the t chromosomes. Alleles within each group are similar if not identical, although they are borne by chromosomes that have been separated in time and space. The presence of similar alleles in Mus musculus and Mus domesticus suggests that some of them may have been separated for more than 1 million years. This must also be the minimal age of the t chromosomes but, because at least two of the three main branches appear to be related in their origin, the actual age of t chromosomes could be much greater. The observations support the proposal that H-2 genes evolve slowly.
The translocated c-myc oncogene of Raji Burkitt lymphoma cells is not expressed in human lymphoblastoid cells.
Authors: Nishikura K , Erikson J , ar-Rushdi A , Huebner K , Croce C M
Published: May 2006

We hybridized Raji Burkitt lymphoma cells, which carry a t(8;14) chromosome translocation, with human lymphoblastoid cells to study the expression of the translocated cellular myc oncogene (c-myc) in the hybrid cells. In Raji cells the c-myc oncogene is translocated to a switch region of the gamma heavy chain locus (S gamma). Because of sequence alterations in the 5' exon of the translocated c-myc oncogene in this cell line, it is possible to distinguish the transcripts of the translocated c-myc gene and of the normal c-myc gene. S1 nuclease protection experiments with a c-myc first exon probe indicate that Raji cells express predominantly the translocated c-myc gene, while the level of expression of the normal c-myc gene is less than 2% of that of the translocated c-myc gene. Somatic cell hybrids between Raji and human lymphoblastoid cells retain the lymphoblastoid phenotype and express only the normal c-myc oncogene. This result indicates that the activation of a c-myc oncogene translocated to a S region depends on the stage of B-cell differentiation of the cells harboring the translocated c-myc gene and not on alterations in the structure of the translocated c-myc oncogene.
D-Tryptophan-6 analog of luteinizing hormone-releasing hormone as a protective agent against testicular damage caused by cyclophosphamide in baboons.
Authors: Lewis R W , Dowling K J , Schally A V
Published: May 2006

Possible protective effects of the agonist [D-Trp6]LH-RH (the D-tryptophan-6 analog of luteinizing hormone-releasing hormone) against testicular damage caused by cyclophosphamide (Cytoxan) were investigated in subhuman primates. Three adult male baboons (Papio anubis) were first subjected to normal semen evaluation by using electroejaculation. The average baseline count for the animals ranged from 95.7 X 10(6) to 585.7 X 10(6) sperm per ml with 90% normal forms and 85% motility with excellent rapid forward progression. After baseline evaluations, two of the animals were treated with daily subcutaneous injections of 0.5 mg of the agonist [D-Trp6]LH-RH. There was an initial rise in serum testosterone after 1 week, but testosterone fell to castration values at 1 month and continued at these levels during treatment with the agonist. There was also an initial rise in sperm concentration 1 month after treatment was started, but after 2 months the animals were azoospermic. After 13 weeks of therapy with [D-Trp6]LH-RH, these two baboons and a third untreated control animal were given cyclophosphamide at a dose of about 3 mg/kg of body weight per day for 4 months. The two animals pretreated with [D-Trp6]LH-RH, continued to receive this agonist until 1 week after the last dose of Cytoxan. In one of the two baboons treated with Cytoxan and the LH-RH agonist, the white blood count fell below 4000 per microliter, and the dose of Cytoxan had to be reduced to 1.5 mg/kg per day for 12 days. The control animal developed azoospermia after 4 months of treatment with cyclophosphamide, and serum testosterone increased while sperm count decreased. Four weeks after the agonist was stopped, serum testosterone in both animals pretreated with [D-Trp6]LH-RH returned to normal levels. The control animal showed a small amount of nonmotile sperm 2.5 months after cessation of treatment, but after 9 months remains oligospermic with poor sperm motility. In one of the animals treated with LH-RH agonist, semen analysis returned to normal pretreatment values 8 months after withdrawal of treatment. The other animal remains oligospermic 10 months after therapy, but the motility is improving. These preliminary results suggest that treatment with LH-RH agonist might decrease the gonadal damage caused by some chemotherapeutic agents.
Cloning and expression of the Rickettsia prowazekii ADP/ATP translocator in Escherichia coli.
Authors: Krause D C , Winkler H H , Wood D O
Published: May 2006

Cosmid clone banks of Rickettsia prowazekii genomic DNA were established in Escherichia coli and screened for expression of the rickettsial carrier-mediated ADP/ATP translocator. Out of 2700 clones screened, a single clone, designated MOB286, accumulated radioactivity when incubated with [alpha-32P]ATP in 100 mM sodium phosphate buffer. This clone carried a plasmid, pMW286, containing a 9-kilobase-pair insert of rickettsial DNA, as established by DNA X DNA hybridizations. Transformation studies with purified pMW286 established that the ability of E. coli cells to accumulate radioactivity was mediated by the recombinant plasmid. Results from experiments in which [3H]ATP was substituted for [alpha-32P]ATP strongly suggested that the radiolabeled ATP was transported intact. Furthermore, [3H]ATP was incorporated into 10% (wt/vol) trichloroacetic acid-precipitable material in a time-dependent manner. Uptake of ATP was also temperature-dependent, insensitive to atractyloside, N-ethylmaleimide, and dinitrophenol, and specific for ADP and ATP. Efflux of radiolabeled nucleotide was observed in the presence of extracellular ADP or ATP but not AMP and was not observed in the absence of extracellular adenine nucleotides. The successful cloning and expression of the rickettsial ADP/ATP translocator in E. coli will permit better characterization of rickettsial bioenergetics and of the metabolic regulation of obligate intracellular parasitism.
Functional channel formation associated with cytotoxic T-cell granules.
Authors: Young J D , Nathan C F , Podack E R , Palladino M A , Cohn Z A
Published: May 2006

Lymphocyte granules from cytotoxic T-lymphocyte lines A2, A11, and R8 were enriched by subcellular fractionation using a Percoll gradient. Granule-enriched fractions showed potent hemolytic activity in the presence of Ca2+. Isolated granules induced rapid Ca2+-dependent membrane depolarization of J774 macrophage-like cells. When tested in planar bilayers, granules induced the formation of Ca2+-dependent functional ion channels of large conductance steps of 1-6 nS in 0.1 M NaCl. Granule-induced channels were resistant to closing by an increase in transmembrane potential, with few channels shifting to the closed state only at voltages of greater than 70 mV, following a Poisson process. These channels showed poor ion selectivity and were permeable to all monovalent and divalent ions (K+, Na+, Li+, Cl-, Ca2+, Mg2+, Zn2+, Ba2+). Ultrastructural examination of soluble granule proteins incubated for 48 hr at 37 degrees C in the presence of Ca2+ revealed ring-like structures of 150-200 A. Structural and functional channel formation may be involved in cytolysis induced by cytotoxic T lymphocytes.
Hemoglobin Long Island is caused by a single mutation (adenine to cytosine) resulting in a failure to cleave amino-terminal methionine.
Authors: Prchal J T , Cashman D P , Kan Y W
Published: May 2006

Hemoglobin Long Island has two separate amino acid abnormalities of beta-globin structure: an extension of the NH2 terminus by a methionine residue and a histidine-to-proline substitution at the normal second position. The NH2-terminal methionine residue, the translation product of an AUG initiation codon, is present only transiently in nascent proteins. Because of the general biological implications of this abnormality, we investigated the nature of the genetic defect of this mutant. We determined the sequence of the relevant portion of the beta-globin mRNA by means of dideoxynucleotide chain termination of the complementary DNA (cDNA) in which an oligonucleotide complementary to codons 10-17 was used as a primer for reverse transcriptase. A histidine-to-proline substitution was confirmed in the mutant mRNA by identifying an adenine-to-cytosine transversion in the second codon. However, we were unable to find any other abnormality at either the AUG initiation codon or in the 56 bases upstream from the adenine-to-cytosine transversion (encompassing most of the 5' untranslated region of the mutant beta-globin mRNA). Thus, it appears that this single lesion probably interferes with the poorly understood methionine-cleaving mechanism that modulates most of prokaryotic and eukaryotic proteins.
Molecular analysis of a deletion mutant provirus of type I human T-cell lymphotropic virus: evidence for a doubly spliced x-lor mRNA.
Authors: Aldovini A , De Rossi A , Feinberg M B , Wong-Staal F , Franchini G
Published: May 2006

The genome of the human T-cell leukemia/lymphotropic virus type I (HTLV-I) contains a functional gene denominated x-lor that may be important in HTLV-I transformation of human T cells. To study the role of x-lor and other HTLV-I genes in cellular transformation, we obtained a transformed nonproducer human T-cell line containing a single defective HTLV-I provirus (HTLV-I 55/PL). This 7-kilobase provirus had undergone a deletion involving the entire envelope gene and the nonconserved region. The point of the deletion corresponded to the junction of a donor splice site, located between the polymerase gene and the envelope gene (nucleotide 5183), and the acceptor site for the mRNA of the x-lor gene (nucleotide 7302). The juxtaposition of nucleotides 5182 and 7302 brings the initiating methionine codon of the envelope gene immediately 5' to the x-lor region, leaving the DNA sequence in frame for expression of a protein product. This finding suggests that a double splicing mechanism is used to express the x-lor gene, and that the defective provirus 55/PL was generated through the reverse transcription of a partially spliced mRNA. Analysis of the x-lor mRNA of other HTLV-I-transformed cell lines revealed that a double splicing process is commonly used. Furthermore, since 55/PL can be faithfully transmitted and is able to immortalize recipient T cells, we can conclude that the envelope gene is not necessary for in vitro transformation by HTLV-I.
Infectious RNA derived by transcription from cloned cDNA copies of the genomic RNA of an insect virus.
Authors: Dasmahapatra B , Dasgupta R , Saunders K , Selling B , Gallagher T , Kaesberg P
Published: May 2006

RNA transcripts of cloned cDNA of the genomic RNAs of BBV (black beetle virus) are infectious to cultured cells of Drosophila melanogaster. Individual transcripts had approximately 10% of the infectivity of the corresponding authentic virion RNA. Progeny virus resulting from transcript infection was phenotypically indistinguishable from the progenitor virus used to generate the original cDNA forms as judged by sucrose density gradient sedimentation, specific infectivity, plaque morphology, and serology. Although the transcript RNAs used to produce this virus had 20 nonviral bases headed by a capping group at their 5' termini, these 20 bases were absent in the progeny viral RNAs. The cDNA forms, and therefore the resulting transcript RNAs, should be readily modifiable by the techniques of recombinant DNA technology both for viral studies and for the insertion of foreign genes into the viral genome and thus into the host cytoplasm.
Phorbol diesters inhibit enzymatic hydrolysis of diacylglycerols in vitro.
Authors: Chabbott H , Cabot M C
Published: May 2006

The effect of phorbol 12-myristate 13-acetate (PMA) on diacylglycerol lipase activity was examined in rat serum, tissue, and cellular preparations by using di[14C]oleoylglycerol, [3H]palmitoylacetylglycerol, and membrane-resident phospholipase C-generated diacylglycerols as substrates. These experiments were conducted to address whether phorbol esters can mimic diacylglycerols in interacting with enzymes other than protein kinase C. Serum hydrolysis of palmitoylacetylglycerol, assayed by the formation of [3H]palmitic acid, was inhibited by PMA, 4-O-methyl-PMA, or phorbol 12,13-dibutyrate (in order of decreasing potency). The hydrolysis of palmitoylacetylglycerol was inhibited more than 40% by the addition of PMA at a 1:1 molar ratio with substrate. The inhibition resembled the competitive type, with a Ki of approximately 2.7 microM. PMA in the 10-60 microM range also inhibited hydrolysis of palmitoylacetylglycerol by lipases from rat brain microsomes and by homogenates of C3H/10T1/2 mouse fibroblasts. PMA was likewise inhibitory when assayed in an intramembrane enzyme-substrate milieu in which diacylglycerols were generated, in situ, by treatment of [3H]palmitate-labeled cell homogenates with phospholipase C. Collectively, these data demonstrate that PMA, which is now thought to act by mimicry of diacylglycerols, can inhibit the action of diacylglycerol lipase. It is possible that such a mechanism is linked to the multiplicity of responses elicited by phorbol diesters and that other agents may function by means of enzyme interactions (post-phospholipase C) to influence the levels of the cellular diacylglycerol mediators.
A block in initiation of simian virus 40 assembly results in the accumulation of minichromosomes containing an exposed regulatory region.
Authors: Ambrose C , Blasquez V , Bina M
Published: May 2006

The initiation of simian virus 40 assembly is blocked at the nonpermissive temperature in cells infected with the viral capsid protein VP1 mutant tsC219. Greater than 95% of the minichromosomes isolated from these cells are accessible to cleavage by Bgl I and Sph I, which recognize the sequences near the viral replication origin and in the transcription enhancer elements, respectively. The accessibility of the Ori region to Bgl I is considerably reduced when virion assembly is allowed to proceed in tsC219-infected cells at the permissive temperature. A reduced accessibility to Bgl I is also observed for chromatin isolated from cells infected with wt776, the wild-type parental strain of tsC219. For wt776 chromatin, variability to Bgl I sensitivity is observed and this can be correlated to the relative virion-to-chromatin yield. A similar correlation is not apparent for restriction endonucleases that recognize sequences within the coding region of simian virus 40 chromatin. These results, considered together, indicate that, when virion assembly initiation is blocked, nucleosomes are nonrandomly arranged with respect to the viral regulatory sequences. It appears that the open regulatory region in minichromosomes is established during replication and that a protected regulatory region is generated with the onset of virion assembly.
Sensitivity of gap junctional conductance to H ions in amphibian embryonic cells is independent of voltage sensitivity.
Authors: Spray D C , Campos de Carvalho A , Bennett M V
Published: May 2006

In vertebrate embryos gap junctional conductance (gj) is reduced by transjunctional voltage (Vj) and by cytoplasmic acidification; in each case sensitivity is comparable to those of other channels gated by voltage and ligand-receptor binding. We show here that the mechanisms by which Vj and intracellular pH (pHi) gate gj are apparently independent. Partial reduction of gj by lowering pHi neither attenuates nor enhances further reduction by Vj. Certain drugs irreversibly (glutaraldehyde, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) or reversibly (retinoic acid) abolish dependence of gj on pHi without appreciably affecting kinetic properties of voltage dependence or the shape of the steady-state Vj-gj relation. These findings suggest that the mechanisms by which pHi and Vj act on the gap junction are at least partially distinct and presumably involve separate regions of the junctional macromolecules.
Atrial natriuretic factor receptors in rat kidney, adrenal gland, and brain: Autoradiographic localization and fluid balance dependent changes
Authors: Lynch David R. , Braas Karen M. , Snyder Soloman H.
Published: May 2006

Mammalian atria contain natriuretic peptides designated atrial natriuretic factors (ANF). Using
in vitro
autoradiography with
125
I-labeled ANF, we have localized high-affinity (
K
d
= 150 pM) ANF binding sites to the glomeruli of the kidney, zona glomerulosa of the adrenal gland, and choroid plexus of the brain. The numbers of sites in both kidney and adrenal are increased in rats deprived of water; increases are detectable within 72 hr of water deprivation in the kidney and within 24 hr in the adrenal gland. Receptor numbers decline in rats given 2.0% NaCl as drinking water and in diabetic rats. The discrete localizations and dynamic alterations of these receptors suggest that ANF regulates fluid balance through diverse but coordinated effects on receptors in numerous organs including the kidney, adrenal, and brain.
Phosphorylation and dephosphorylation of dihydropyridine-sensitive voltage-dependent Ca2+ channel in skeletal muscle membranes by cAMP- and Ca2+-dependent processes.
Authors: Hosey M M , Borsotto M , Lazdunski M
Published: May 2006

The phosphorylation and dephosphorylation of the dihydropyridine-sensitive Ca2+ channel was studied in transverse-tubule membranes isolated from rabbit skeletal muscle. Exposure of these membranes to either the cAMP-dependent protein kinase or a Ca2+/calmodulin-dependent protein kinase resulted in a rapid phosphorylation of a protein with properties similar to the major component of the skeletal muscle Ca2+ channel. The molecular mass of the phosphoprotein was 140 or 160 kDa, depending on the electrophoretic conditions. The stoichiometry of the phosphorylation was calculated to be 0.4-1.0 mol of phosphate per mol of protein. Neither the rate nor the extent of phosphorylation was affected by dihydropyridines. Limited proteolytic digestion of the protein that had been phosphorylated by either or both protein kinases yielded a single phosphopeptide of approximately equal to 5.4 kDa. The Ca2+-dependent phosphatase calcineurin dephosphorylated the membrane-bound Ca2+ channel that had been previously phosphorylated by either protein kinase. The results suggest that the major component of the dihydropyridine-sensitive Ca2+ channel from skeletal muscle can be effectively phosphorylated and dephosphorylated in its native state by cAMP- and Ca2+-dependent processes.
The active immunoglobulin kappa chain gene is packaged by non-ubiquitin-conjugated nucleosomes.
Authors: Huang S Y , Barnard M B , Xu M , Matsui S , Rose S M , Garrard W T
Published: May 2006

To elucidate the molecular features of active chromatin, we have mapped, by two-dimensional electrophoresis, the protein composition of nucleosomes that package the immunoglobulin kappa chain gene of mouse plasmacytoma cells. Nucleoprotein particles that possess the active kappa chain gene comigrate with bulk mononucleosomes that contain high mobility group proteins HMG-14 or -17 but lack histone H1. High electrophoretic resolution of the underlying core particles, after removal of ubiquitin by isopeptidase treatment, reveals that these nucleosomes are nonubiquitinated, even though they coincidently migrate with bulk ubiquitinated particles. This distinctive electrophoretic behavior may be correlated with the presence of histone H2A.X. Nucleosomes exhibiting these unusual properties appear to span at least 10 kilobases, in both transcribed and nontranscribed regions, suggesting that mechanisms independent of transcription exist to initiate, maintain, and propagate a common chromatin phenotype over long distances along the kappa chain locus.