bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA gyrase binds to the family of prokaryotic repetitive extragenic palindromic sequences.
Authors: Yang Y , Ames G F
Published: May 2006

A family of repetitive extragenic palindromic (REP) sequences is composed of hundreds of copies distributed throughout the chromosome. Their palindromic nature and conservation suggested that they are specifically recognized by a protein(s). We have identified DNA gyrase [DNA topoisomerase (ATP-hydrolysing), EC 5.99.1.3] as one of the REP-binding proteins. Gyrase has at least a 10-fold higher affinity for DNA containing REP sequences than for DNA not containing REP sequences. Binding effectiveness correlates directly with the number of REP sequences in the DNA. DNase I footprinting shows that gyrase protects 205 base pairs on a REP-containing DNA fragment enclosing the REP sequences. In agreement with the above results, a comparison of the REP consensus sequence with the sequence of previously identified pBR322 "strong" gyrase cleavage sites reveals a high degree of homology. Because REP sequences are numerous and found throughout the genome, we suggest they have physiological functions mediated through their interaction with gyrase, such as being sites of action for the maintenance of DNA supercoiling. In addition, we speculate that these interactions may be of a structural nature, such as involvement in the higher-order structure of the bacterial chromosome.
Dual mechanism of repression at a distance in the lac operon.
Authors: Flashner Y , Gralla J D
Published: May 2006

The mechanism by which the internal lacZ gene sequence O2 influences lac repression was investigated by using in vivo footprinting of operon mutants. Quantitative in vivo binding curves show that O2 strengthens by approximately 3-fold repressor binding to O1 that is located 400 base pairs upstream at the transcription start site. The internal O2 sequence also contributes to repression by a second mechanism: repressor bound internally blocks elongation of beta-galactosidase gene expression. This secondary mechanism of repression is facilitated by the remote O1 operator that strengthens binding to O2 12-fold. Thus, lac repression involves two mechanisms, both of which involve cooperation between remote operator elements. During mild repression only the initiation mechanism applies, but more severe repression favors formation of the presumptive O1-O2 repression loop that allows both mechanisms to act simultaneously.
Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene.
Authors: Efrat S , Linde S , Kofod H , Spector D , Delannoy M , Grant S , Hanahan D , Baekkeskov S
Published: May 2006

Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The cells produce both proinsulin I and II and efficiently process each into mature insulin, in a manner comparable to normal beta cells in isolated islets. Electron microscopy reveals typical beta-cell type secretory granules, in which insulin is stored. Insulin secretion is inducible up to 30-fold by glucose, although with a lower threshold for maximal stimulation than that for normal beta cells. beta TC lines can be repeatedly derived from primary beta-cell tumors that heritably arise in the transgenic mice. Thus, targeted expression of an oncogene with a cell-specific regulatory element can be used both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype.
Analysis of pivotal-differential evolutionary patterns
Authors: Kimber Gordon , Yen Yang
Published: May 2006

The numerical analysis of meiosis in hybrids between a wild allotetraploid and an autotetraploid of one of its putative diploid progenitors allows the identification of which genomes are pairing and also the verification of pivotal-differential evolution. This type of analysis should be applicable to all genera in which allopolyploid series exist.
Intrachromosomal rearrangements mediated by hobo transposons in Drosophila melanogaster.
Authors: Lim J K
Published: May 2006

The recurring intrachromosomal rearrangements observed in an unstable X chromosome, designated Uc, of Drosophila melanogaster are shown to be mediated by hobo transposable elements. Each of 29 chromosome rearrangement breakpoints in 16 gross aberrations detected in the Uc-derived X chromosomes had a hobo element. In one particular unstable X chromosome line selected for detailed studies, a hobo element was found in each of the five hot spots for rearrangements. Furthermore, hobo elements at deletion hot spots were found to lie in the same orientation, whereas those hobo elements at inversion hot spots were in the opposite orientation. The restriction maps of two phage lambda clones containing rearrangement breakpoints indicated that a hobo element was inserted exactly at the breakpoints. Pairing of hobo elements in the same chromosome followed by recombination between the paired hobo elements is suggested as the explanation for the intrachromosomal aberrations observed in the Uc X chromosomes. A clear qualitative difference among the hobo elements in their ability to participate in rearrangement formation was noted. It was also found that each of the 11 recessive lethal mutations mapped in the 6F1-2 doublet had a hobo element in the doublet, whereas none of the 16 independent revertants of the mutation had a hobo element in the site. This observation indicates that hobo movement is responsible for production and subsequent instability of recessive lethal mutations in the 6F region of the Uc X chromosomes.
Formation of IgE-binding factors by T cells of patients infected with human immunodeficiency virus type 1.
Authors: Carini C , Margolick J , Yodoi J , Ishizaka K
Published: May 2006

Peripheral blood mononuclear cells of 10 out of 26 patients with human immunodeficiency virus type 1 (HIV-1) released IgE-binding factors, as determined by two independent assays. The formation of the factors by the mononuclear cells was enhanced by incubation of the cells with homologous IgE. In the presence of IgE, peripheral blood mononuclear cells from 15 of the 26 patients formed a detectable amount of IgE-binding factors, whereas those of normal individuals of allergic rhinitis patients failed to do so. The major source of IgE-binding factors was the T cells of the HIV-1-infected patients. The CD8+ T cells from a HIV-1-infected patient formed IgE-binding factors upon incubation with IgE, and type II receptors for Fc epsilon were detected on both CD4+ T cells and CD8+ T cells in one of five patients studied. It was also found that culture supernatants of mononuclear cells from HIV-1-infected patients released soluble factors that induce normal human T cells to form IgE-binding factors. The results suggest that lymphocytes of some HIV-1-infected patients are activated to produce lymphokines regulating formation of IgE-binding factors.
Suppression and induction of epileptic activity by neuronal grafts.
Authors: Buzsáki G , Ponomareff G , Bayardo F , Shaw T , Gage F H
Published: May 2006

Fetal rat brain cell suspensions prepared from either the locus coeruleus region or hippocampus were implanted bilaterally into the subcortically denervated seizure-prone hippocampus of adult rats. Animals with locus coeruleus grafts were protected against picrotoxin-induced behavioral seizures and had significantly fewer interictal spikes. In contrast, in rats with fetal hippocampal grafts the incidence of interictal spikes was significantly higher than in lesion-only controls, and spontaneous behavioral seizures occurred in almost half of the animals. We suggest that neuronal grafting offers an alternative method for studying the mechanisms and control of epileptic brain activity.
Long-term sensitization in Aplysia increases the number of presynaptic contacts onto the identified gill motor neuron L7.
Authors: Bailey C H , Chen M
Published: May 2006

We have used the gill and siphon withdrawal reflex of Aplysia to study the morphological basis of the persistent synaptic plasticity that underlies long-term sensitization. One critical locus for storage of the memory for sensitization is the set of monosynaptic connections between identified siphon sensory neurons and gill and siphon motor neurons. To complement previous morphological studies of the presynaptic terminals of identified sensory neurons, we examined the effects of long-term sensitization on the structure of an identified postsynaptic target--the gill motor neuron L7. We found an increase in the frequency, size, and vesicle complement of presynaptic contacts onto L7 processes in sensitized compared to control animals. Combined, these data indicate a striking increase in the percentage of the surface area of L7 that is occupied by synaptic contacts after long-term training. These results are consistent with our observations that sensitization produces an increase in the synapses that the sensory neurons make on their target cells and provide additional support for the hypothesis that changes in synapse number may represent a mechanism underlying long-term memory.
The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit.
Authors: Kilimann M W , Zander N F , Kuhn C C , Crabb J W , Meyer H E , Heilmeyer L M
Published: May 2006

We have cloned cDNA molecules encoding the beta subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature beta subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the alpha subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the beta subunit and all seven phosphorylation sites known for the alpha subunit are located in these unique domains.
Secretion in yeast of human lysozymes with different specific activities created by replacing valine-110 with proline by site-directed mutagenesis.
Authors: Kikuchi M , Yamamoto Y , Taniyama Y , Ishimaru K , Yoshikawa W , Kaisho Y , Ikehara M
Published: May 2006

Computer graphics indicate that a steric hindrance exists between valine-110 side chain of human lysozyme (EC 3.2.1.17) and an acetyl group of a modified substrate that contains N6,O-diacetylmuramic acid. To alter the substrate specificity of human lysozyme to be effective on the modified substrate, we replaced the valine-110 residue with various amino acids by site-directed mutagenesis. One of the mutant proteins (valine residue replaced with proline:P110) was secreted in Saccharomyces cerevisiae as at least four components (P110-A, P110-B, P110-C, and P110-D) with different specific activities. Two components, P110-B and P110-D, were isolated in a pure form and structurally characterized. The results suggest that this mutation lowered the lytic activity against Micrococcus lysodeikticus by changing a local conformation of the catalytic site while keeping almost the same substrate binding sites. Our results also indicate that cis/trans isomerization of prolyl peptide bonds probably occurs in vivo and that the conformational change of protein as well as point mutations in genes might influence the molecular evolution of the protein.
Familial defective apolipoprotein B-100: enhanced binding of monoclonal antibody MB47 to abnormal low density lipoproteins.
Authors: Weisgraber K H , Innerarity T L , Newhouse Y M , Young S G , Arnold K S , Krauss R M , Vega G L , Grundy S M , Mahley R W
Published: May 2006

Familial defective apolipoprotein (apo) B-100 is a recently described genetic disorder that appears to result from a mutation in the apoB-100 gene. This disorder is characterized by hypercholesterolemia resulting from elevated plasma concentrations of low density lipoprotein LDL. The disorder was first detected in three members of one family. The LDL from affected subjects binds defectively (approximately 30% of normal) to LDL receptors, retarding the clearance of LDL from plasma. In the present study, two other members of the affected family were found to possess abnormal LDL. In addition, abnormal LDL with a similar binding defect were found in a second, unrelated family. In both families, the defect is transmitted over three generations as an autosomal codominant trait and all affected members are heterozygotes. Since there is only one apoB-100 molecule per LDL particle, the abnormal LDL in heterozygous subjects is made up of two populations of particles: one that has normal binding activity to receptors and one that binds defectively. To localize the mutation in apoB-100, the binding of five apoB-100-specific monoclonal antibodies to abnormal LDL was assessed in a solid-phase RIA. Only antibody MB47, whose epitope is between residues 3350 and 3506, distinguished abnormal LDL from normal LDL isolated from control subjects with normal lipid levels; MB47 bound with a higher affinity (by approximately 60%) to abnormal LDL. In every individual with abnormal LDL, the MB47 antibody bound with a higher affinity. The convenience of this assay will facilitate screening of large populations to determine the frequency of this disorder.
Molecular cloning, sequence analysis, and expression in Escherichia coli of the cDNA for guanidinoacetate methyltransferase from rat liver.
Authors: Ogawa H , Date T , Gomi T , Konishi K , Pitot H C , Cantoni G L , Fujioka M
Published: May 2006

Five cDNA clones encoding rat liver guanidinoacetate methyltransferase (S-adenosyl-L-methionine: guanidinoacetate N-methyltransferase, EC 2.1.1.2) were isolated from a lambda gt11 cDNA library by use of a polyclonal antibody to the purified enzyme. Sequence analysis of the longest cDNA indicated that it consisted of 711 base pairs (bp) of coding region, 51 bp of 5' noncoding region, and 162 bp of 3' noncoding region excluding the poly(A) tail. The amino acid sequence deduced from the cDNA contained the sequences of NH2-terminal and three tryptic peptides. The predicted amino acid composition and molecular weight were in excellent agreement with those obtained with the purified enzyme. Introduction of the cDNA into plasmid pUC118 having the lac promoter resulted in a production in Escherichia coli of a Mr 26,000 polypeptide in the presence of isopropyl beta-D-thiogalactopyranoside. This protein represented as much as 5% of the bacterial soluble protein and showed the guanidinoacetate methyltransferase activity. Sequence analysis and tryptic peptide mapping indicated that the enzyme obtained by the recombinant DNA procedures was structurally identical to the liver enzyme, except for the absence of the NH2-terminal blocking group. Also, the enzyme showed kinetic properties indistinguishable from those of the liver enzyme.
Glucocorticoid-dependent complementation of a hepatoma cell variant defective in viral glycoprotein sorting.
Authors: John N J , Bravo D A , Haffar O K , Firestone G L
Published: May 2006

We have utilized the rat hepatoma (HTC) cell sorting variant CR4 to examine the glucocorticoid-regulated pathways that localize mouse mammary tumor virus glycoproteins to the cell surface. The defective sorting of cell surface mouse mammary tumor virus glycoproteins in CR4 cells was complemented after fusion with either normal rat hepatocytes or uninfected HTC cells. Indirect immunofluorescence of transient heterokaryons revealed that the regulated localization of mouse mammary tumor virus glycoproteins was dependent upon glucocorticoid treatment and required de novo RNA and protein synthesis. Thus, a glucocorticoid-regulated trafficking activity, unrelated to mouse mammary tumor virus sequences, which is induced in both adult rat liver and cultured hepatoma cells, can act in trans to mediate an intracellular sorting pathway for membrane glycoproteins.
Differential expression of genes in potato tubers after wounding
Authors: Logemann J. , Mayer J. E. , Schell J. , Willmitzer L.
Published: May 2006

Tubers of a common tetraploid species of
Solanum tuberosum
(Granola) were mechanically wounded by slicing. After 18 hr only small differences were detectable in the pattern of the steady-state protein extracted from wounded versus unwounded tubers. In contrast the protein pattern obtained by
in vitro
translation of mRNA isolated from wounded and unwounded tubers differed significantly. A cDNA library was established and screened for wound-induced cDNA clones by differential hybridization. Two clones, wun-1 and wun-2, were found that corresponded to genes that were highly expressed in wounded potato tubers but were not expressed in unwounded tubers. The expression of the gene corresponding to wun-1 is detectable 30 min after wounding; the expression of the gene corresponding to wun-2 is detectable 4 hr after wounding. The expression of both genes (hereafter referred to as wun-1 and wun-2) remains constant for up to 24 hr after wounding. Interestingly the RNA corresponding to patatin, a major storage protein of potato tubers, behaves in the opposite way; it decreases dramatically in tubers within 30 min after wounding. The low level of patatin mRNA observed in unwounded roots and stems also disappears after wounding. Run-off transcription experiments, performed with isolated nuclei, indicate that the activation of the wound-induced genes as well as the inhibition of the patatin gene are controlled at the transcriptional level.
Structural conservation among three homologous introns of bacteriophage T4 and the group I introns of eukaryotes.
Authors: Shub D A , Gott J M , Xu M Q , Lang B F , Michel F , Tomaschewski J , Pedersen-Lane J , Belfort M
Published: May 2006

Three group I introns of bacteriophage T4 have been compared with respect to their sequence and structural properties. The introns include the td intervening sequence, as well as the two newly described introns in the nrdB and sunY genes of T4. The T4 introns are very closely related, containing phylogenetically conserved sequence elements that allow them to be folded into a core structure that is characteristic of eukaryotic group IA introns. Similarities extend outward to the exon sequences surrounding the three introns. All three introns contain open reading frames (ORFs). Although the intron ORFs are not homologous and occur at different positions, all three ORFs are looped-out of the structure models, with only the 3' ends of each of the ORFs extending into the secondary structure. This arrangement invites interesting speculations on the regulation of splicing by translation. The high degree of similarity between the T4 introns and the eukaryotic group I introns must reflect a common ancestry, resulting either from vertical acquisition of a primordial RNA element or from horizontal transfer.
Metallothionein mRNA expression in mice homozygous for chromosomal deletions around the albino locus.
Authors: DeFranco D , Morris S M , Leonard C M , Gluecksohn-Waelsch S
Published: May 2006

Deletions in chromosome 7 of the mouse affect the expression of the metallothionein gene Mt-1, which maps on chromosome 8, and steady-state levels of Mt-1 mRNA are reduced to 15-40% of normal in livers of newborn mice homozygous for either the c3H or c14CoS deletion. Glucocorticoids fail to induce hepatic Mt-1 mRNA levels in deletion homozygotes in contrast to normal littermates. However, zinc chloride is effective in inducing Mt-1 mRNA levels in livers of deletion homozygotes as well as of their normal littermates. Other tissues (e.g., kidney and intestine) of deletion homozygotes express basal levels of Mt-1 mRNA higher than those of normal littermates. In the intestine these are furthermore inducible by both hormonal and metal agents. Thus, loss of inducibility of the Mt-1 gene in deletion homozygotes concerns glucocorticoids only and is furthermore restricted to specific cell types (i.e., hepatocytes). The trans-acting factor(s) normally encoded in the deleted region of chromosome 7 appears to be instrumental in conferring on the metallothionein gene in hepatocytes the essential competence to respond to hormonal inducing signals.
Antigen presentation to HLA class II-restricted measles virus-specific T-cell clones can occur in the absence of the invariant chain.
Authors: Sekaly R P , Jacobson S , Richert J R , Tonnelle C , McFarland H F , Long E O
Published: May 2006

A human fibroblast line expressing HLA-DR1 antigen on its surface was generated by transfection with DR alpha and DR beta cDNAs. Expression of the invariant chain gene was not detectable in the transfected fibroblasts and was not induced by infection with measles virus. Lysis of measles virus-infected cells occurred with DR1- but not with DR4-restricted measles virus-specific cytotoxic T-lymphocyte (CTL) clones and was inhibited by a monoclonal antibody specific for DR antigen. Therefore, the invariant chain is not required for DR-restricted presentation of measles virus antigens by this fibroblast line. Transfected fibroblasts were lysed as efficiently as an autologous B-cell line even though they expressed much less surface DR antigen. Lysis of both the transfected fibroblasts and the B-cell line was insensitive to treatment with chloroquine. These results demonstrate that expression of a DR alpha beta heterodimer at the surface of this fibroblast line is necessary and sufficient for presentation of measles virus antigens to specific CTL clones.
Differential regulation of amyloid-beta-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease.
Authors: Higgins G A , Lewis D A , Bahmanyar S , Goldgaber D , Gajdusek D C , Young W G , Morrison J H , Wilson M C
Published: May 2006

We have mapped the neuroanatomical distribution of amyloid-beta-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-beta-protein mRNA appears to be expressed in all hippocampal neurons, but at different levels of abundance. In the central nervous system of monkey and normal aged human, image analysis shows that neurons of the dentate gyrus and cornu Ammonis fields contain a 2.5-times-greater hybridization signal than is present in neurons of the subiculum and entorhinal cortex. In contrast, in the Alzheimer disease hippocampal formation, the levels of amyloid-beta-protein mRNA in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-beta-protein gene expression may be altered in Alzheimer disease.
CheA protein, a central regulator of bacterial chemotaxis, belongs to a family of proteins that control gene expression in response to changing environmental conditions.
Authors: Stock A , Chen T , Welsh D , Stock J
Published: May 2006

During bacterial chemotaxis, the binding of stimulatory ligands to chemoreceptors at the cell periphery leads to a response at the flagellar motor. Three proteins appear to be required for receptor-mediated control of swimming behavior, the products of the cheA, cheW, and cheY genes. Here we present the complete nucleotide sequence of the Salmonella typhimurium cheA gene together with the purification and characterization of its protein product. The protein is a 73,000 Mr cytoplasmic constituent. Amino acid-sequence comparisons indicate that it belongs to a family of bacterial regulatory proteins including the products of the cpxA, dctB, envZ, ntrB, phoR, phoM, and virA genes. Each member of this family has a conserved domain of approximately equal to 200 residues within its C terminus. We have previously shown that another chemotaxis protein, CheY, represents a domain of protein structure that has been conserved within a second large family of bacterial regulatory proteins. Each protein of the CheA family seems to function as a regulator of a different CheY homologue. Although each pair of proteins appears to produce a specialized response to a distinct type of stimulus, the relationships in primary structure suggest that a similar molecular mechanism may be involved.
Electron spin resonance of charge carriers in chlorophyll a/water micelles
Authors: Bowman M. K. , Michalski T. J. , Tyson R. L. , Worcester D. L. , Katz J. J.
Published: May 2006

Chlorophyll a/water micelles (P740) prepared in hydrocarbon media have been shown by small-angle neutron scattering to consist of hollow cylinders whose surface is formed of a monolayer of chlorophyll crosslinked by water. The micelles can be reversibly oxidized or reduced to generate highly mobile holes or electrons that undergo rapid, one-dimensional transport along the chains of chlorophyll macrocycles comprising the surface of the micelles. Large π-π overlap within the chains facilitates the one-dimensional charge transport and is expected to do the same for energy transport. Structural defects in the micelle surface act as boundaries for charge transport, confining the spins to one-dimensional domains of approximately 200 macrocycles. The one-dimensional transport within the limited domains results in motionally narrowed electron spin resonance lines with some residual inhomogeneous broadening. Although the chlorophyll a incorporated in micelles is more easily oxidized than is monomeric chlorophyll a, it is much more resistant to chemical alteration.
Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes.
Authors: Farr C J , Saiki R K , Erlich H A , McCormick F , Marshall C J
Published: May 2006

In vitro DNA amplification followed by oligonucleotide dot blot analysis were used to study RAS gene mutations in acute myeloid leukemia (AML). Fifty-two presentation AML DNAs were screened for mutations in codons 12, 13, and 61 of NRAS and in codons 12 and 61 of KRAS and HRAS. Fourteen (27%) contained mutations--all in NRAS and predominantly in codon 12. The most common amino acid substitution identified was of glycine by aspartic acid at codon 12 (7/18), with a G----A transition being the most common base change (11/18). No particular correlation was observed between disease subtype and the incidence or type of NRAS mutation. In DNA samples from four patients, 2 NRAS mutations were found to coexist. NIH 3T3 focus-formation assays revealed that in each case the mutations were present in different NRAS alleles. We also report the absence of a mutated RAS gene in relapse DNAs of four patients in which a RAS oncogene had been detected at presentation. These observations suggest that RAS mutations arise as part of the evolution of neoplastic transformation.
Highly potent antagonists of luteinizing hormone-releasing hormone free of edematogenic effects.
Authors: Bajusz S , Kovacs M , Gazdag M , Bokser L , Karashima T , Csernus V J , Janaky T , Guoth J , Schally A V
Published: May 2006

To eliminate the undesirable edematogenic effect of the luteinizing hormone-releasing hormone (LH-RH) antagonists containing basic D amino acids at position 6, exemplified by [Ac-D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH [Phe(pCl) indicates 4-chlorophenylalanine], analogs with D-ureidoalkyl amino acids such as D-citrulline (D-Cit) or D-homocitrulline (D-Hci) at position 6 were synthesized and tested in several systems in vitro and in vivo. HPLC analysis revealed that the overall hydrophobicity of the D-Cit/D-Hci6 analogs was similar to that of the basic D-Arg6 antagonists. In vitro, most of the analogs completely inhibited LH-RH-mediated luteinizing hormone release in perfused rat pituitary cell systems at an antagonist to LH-RH molar ratio of 5:1. In vivo, the most active peptides, [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Cit6,D-Ala10]LH-RH [Nal(2) indicates 3-(2-naphthyl)alanine] and its D-Hci6 analog, caused 100% inhibition of ovulation in cycling rats in doses of 3 micrograms and suppressed the luteinizing hormone level in ovariectomized female rats for 47 hr when administered at doses of 25 micrograms. Characteristically, these peptides did not exert any edematogenic effects even at 1.5 mg/kg. These properties of the D-Cit/D-Hci6 antagonists may make them useful clinically.
Senescent erythrocytes: isolation of in vivo aged cells and their biochemical characteristics.
Authors: Suzuki T , Dale G L
Published: May 2006

Rabbit erythrocytes were covalently labeled with biotin and then infused into the donor animal. Up to 60 days after infusion, the biotinylated cells were selectively isolated by their affinity for avidin. These aged erythrocytes, which are within 10 days of death, were analyzed for several biochemical parameters. The 2,3-bisphosphoglycerate and glutathione levels of these cells were constant with age; however, the adenosine 5'-triphosphate concentrations increased approximately 75% as the cells approached the end of their life span. The activities of several glycolytic enzymes did not change with age.
Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells.
Authors: Haycock J W , Browning M D , Greengard P
Published: May 2006

Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32PO4, exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a Mr approximately equal to 100,000 protein and a Mr approximately equal to 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in naDodSO4/polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins ("100-kDa," "87-kDa," and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. "100-kDa" is a Mr approximately equal to 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, "87-kDa" is a Mr approximately equal to 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of Mr approximately equal to 74,000 (IIIa) and Mr approximately equal to 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. Furthermore, 100-kDa was shown to be identical to the Mr approximately equal to 100,000 protein whose phosphorylation was increased by acetylcholine treatment. The acetylcholine-dependent increase in phosphorylation of tyrosine hydroxylase, 100-kDa, 87-kDa, and protein III required extracellular calcium and was mimicked by nicotine, veratridine, elevated K+, and calcium ionophore A23187, but not by muscarine. In addition, forskolin increased the phosphorylation of tyrosine hydroxylase, 100-kDa, and protein III, but not that of 87-kDa. Phorbol 12,13-dibutyrate increased the phosphorylation of tyrosine hydroxylase, 87-kDa, and protein III, but not that of 100-kDa. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects.
DNA bending is induced in an enhancer by the DNA-binding domain of the bovine papillomavirus E2 protein.
Authors: Moskaluk C , Bastia D
Published: May 2006

The E2 gene of bovine papillomavirus type 1 has been shown to encode a DNA-binding protein and to trans-activate the viral enhancer. We have localized the DNA-binding domain of the E2 protein to the carboxyl-terminal 126 amino acids of the E2 open reading frame. The DNA-binding domain has been expressed in Escherichia coli and partially purified. Gel retardation and DNase I "footprinting" on the bovine papillomavirus type 1 enhancer identify the sequence motif ACCN6GGT (in which N = any nucleotide) as the E2 binding site. Using electrophoretic methods we have shown that the DNA-binding domain changes conformation of the enhancer by inducing significant DNA bending.
An epistatic mating system model can produce permanent cytonuclear disequilibria in a hybrid zone.
Authors: Arnold J , Asmussen M A , Avise J C
Published: May 2006

We examine the evolutionary dynamics of gametic and genotypic disequilibria between a cytoplasmic gene and a nuclear gene under two mating system models relevant to hybrid zones. In the first model, in which female mating preference is determined by an epistatic interaction between the two loci, permanent nonzero cytonuclear disequilibria are possible for a variety of initial genotype frequencies, particularly when rates of assortative mating for the two parental species are high. In contrast, when mating preference is effectively determined by interaction between a cytoplasmic gene and the multilocus nuclear genotypes characteristic of the parental species, all cytonuclear disequilibria, as well as frequencies of pure parentals, rapidly decay to zero unless assortative mating is nearly perfect. Results of the models are applied to the interpretation of observed cytonuclear associations in a hybrid population of Hyla tree frogs.
The effect of quaternary structure on the kinetics of conformational changes and nanosecond geminate rebinding of carbon monoxide to hemoglobin.
Authors: Murray L P , Hofrichter J , Henry E R , Ikeda-Saito M , Kitagishi K , Yonetani T , Eaton W A
Published: May 2006

To determine the effect of quaternary structure on the individual kinetic steps in the binding of carbon monoxide to the alpha subunit of hemoglobin, time-resolved absorption spectra were measured after photodissociation of carbon monoxide from a hemoglobin tetramer in which cobalt was substituted for iron in the beta subunits. Cobalt porphyrins do not bind carbon monoxide. Spectra were measured in the Soret region at room temperature after time delays that varied from a few nanoseconds to the completion of ligand rebinding at about 100 ms. The results show that the liganded molecule, alpha(Fe-CO)2 beta(Co)2, is in the R state, but can be almost completely switched into the T state by the allosteric effectors inositol hexaphosphate and bezafibrate. The geminate yield, which is the probability that the ligand rebinds to the heme from within the protein, is found to be 40% for the R state and less than 1% for the T state. According to the simplest kinetic model, these results indicate that carbon monoxide enters the protein in the R and T quaternary conformations at the same rate, and that the 60-fold decrease in the overall binding rate, of carbon monoxide to the alpha subunit in the T state compared to the R state is almost completely accounted for by the decreased probability of binding after the ligand has entered the protein. The results further suggest that the low probability for the T state results from a decreased binding rate to the heme and not from an increased rate of return of the ligand to the solvent.
Isolation of cDNAs encoding desmosomal plaque proteins: evidence that bovine desmoplakins I and II are derived from two mRNAs and a single gene.
Authors: Green K J , Goldman R D , Chisholm R L
Published: May 2006

Desmoplakins (DPs) I and II (approximately equal to 240 and approximately equal to 210 kDa) are major components of the internal portion of the desmosomal cytoplasmic plaque. Desmosomes play a crucial role in cell-cell adhesion and serve as specific attachment sites for cytoplasmic intermediate filaments. Although DP-I and -II are closely related molecules, their structure (i.e., amino acid or DNA sequence) has not been determined. In addition, it is not known whether these proteins are derived from one or more genes or whether they result from posttranscriptional or posttranslational events. This paper describes the isolation and characterization of eight DP cDNA clones from a bovine lambda gt11 expression library. Fusion proteins from six of these clones selected antibodies that reacted with DP-I and -II and two selected antibodies that reacted with DP-I alone. Antibodies made against fusion protein produced by the DP1A clone reacted specifically with DP-I and -II on immunoblots. When used for indirect immunofluorescence on bovine tongue cryostat sections and cultured mouse keratinocytes, these antibodies produced a typical desmosomal staining pattern. RNA blot analysis demonstrated hybridization of three DP-I/II cDNA probes with two messages of approximately equal to 7.5 and approximately equal to 9.5 kilobases in bovine tongue RNA. In contrast, a cDNA clone that affinity-purified antibodies reacting with DP-I only hybridized exclusively with the 9.5-kilobase band. Southern blots of genomic DNA digested with a panel of restriction enzymes were hybridized with one probe derived from a DP-I/II clone and with one from a DP-I clone. Both probes hybridized with single bands of the same size in each digested sample of DNA. Together, these data suggest that DP-I and DP-II are translated from two separate messages in bovine tongue and that these messages may be derived from a single gene.
Mitotic sectored colonies: evidence of heteroduplex DNA formation during direct repeat recombination.
Authors: Ronne H , Rothstein R
Published: May 2006

In yeast meiosis, ascosporal colonies are sometimes sectored for a marker--i.e., half the colony has one allele and half has the other. This is interpreted as replicative resolution of heteroduplex DNA (hDNA) formed as a recombination intermediate. We have looked for similar evidence of hDNA formation during mitotic recombination between two repeated sequences on the same chromosome. The two repeats, an ochre suppressor and a wild-type tRNA gene, are separated by plasmid DNA and the URA3 marker. Recombination between the repeats excises the URA3 gene and one copy of the repeat, leaving either the wild-type tRNA or the suppressor on the chromosome. A red/white color assay is used to distinguish between the two. We find that some colonies that have lost the URA3 gene are sectored for the suppressor. This suggests that hDNA is formed across the anticodon during the recombination event and then resolved by replication. The disruption of either of two genes involved in recombination and repair, RAD1 and RAD52, does not significantly alter the frequency of sectored colony formation during plasmid excision.
Analysis of a DNase I-hypersensitive site in transgenic Drosophila reveals a key regulatory element of Sgs3.
Authors: Ramain P , Giangrande A , Richards G , Bellard M
Published: May 2006

We have undertaken chromatin studies on transformed Drosophila strains carrying DNA sequences modified in the region of the DNase I (EC 3.1.4.5)-hypersensitive sites -750 and -600 base pairs upstream from the Sgs3 start site. Although both sites are developmentally specific, modifications in the -750 site have little or no effect on Sgs3-encoded transcript levels, whereas either deletion or replacement of sequences at the -600 site causes an important reduction in transcript levels. The element associated with the -600 site enhances Sgs3 transcription when displaced with respect to the start site. This combined approach has defined sequence elements necessary both for normal transcript levels as well as the chromatin structure characteristic of Sgs3 activity in vivo.
Helper T-cell clones that recognize autologous insulin are stimulated in nonresponder mice by pork insulin.
Authors: Whiteley P J , Jensen P E , Pierce C W , Abruzzini A F , Kapp J A
Published: May 2006

Murine antibody responses to various species of insulin are under major histocompatibility complex-linked Ir gene control. Beef insulin differs from pork insulin by only two amino acids in the A-chain loop, yet strain C57BL/10 (B10) mice produce insulin-specific antibodies after immunization with beef insulin and fail to produce antibody after stimulation with pork insulin. Nevertheless, pork insulin primes helper T cells in B10 mice that can be demonstrated if insulin-specific Lyt-1-, -2+ suppressor T cells are removed. Not only do the pork insulin-primed helper and suppressor T cells cross-react with autologous insulin, but also rat insulin (the amino acid sequence of which is identical to mouse insulin) elicits functionally identical helper and suppressor T cells. In this report, we demonstrate that in B10 mice the frequency of helper T cells stimulated by pork insulin is equivalent to that stimulated by beef insulin and that helper T-cell clones induced by beef and pork insulin are major histocompatibility complex-restricted T cells that proliferate, produce lymphokines, and provide helper activity after activation. These helper T-cell clones exhibit different antigenic fine specificities: beef insulin-induced clones respond to beef insulin but not pork or autologous insulin, whereas pork insulin-induced clones cross-react with all species of insulin tested, including rat insulin. In addition, the helper activity of cloned pork insulin-specific T cells is abrogated by pork insulin-primed suppressor T cells. These data support the hypotheses that Ir gene control of antibody responses to certain antigens involves mechanisms used for maintenance of self-tolerance.
Characterization of a thyroid hormone receptor expressed in human kidney and other tissues.
Authors: Nakai A , Seino S , Sakurai A , Szilak I , Bell G I , DeGroot L J
Published: May 2006

A cDNA encoding a specific form of thyroid hormone receptor expressed in human liver, kidney, placenta, and brain was isolated from a human kidney library. Identical clones were found in human placenta and HepG2 cDNA libraries. The cDNA encodes a 490-amino acid protein (Mr, 54,824). When expressed and translated in vitro, the protein product binds triiodothyronine with Ka of 2.3 X 10(9) M-1. This protein, designated human thyroid hormone receptor type alpha 2 (hTR alpha 2), has the same domain structure as other members of the v-erbA-related superfamily of receptor genes. It is similar to thyroid hormone receptor type alpha described in chicken and rat and less similar to human thyroid hormone receptor type beta (formerly referred to as c-erbA beta) from placenta. However, it is distinguished from these receptors by an extension of the C-terminal hormone binding domain making it 80 amino acids longer than rat thyroid hormone receptor type alpha 1 [Thompson, C.C., Weinberger, C., Lebo, R. & Evans, R. M. (1987) Science 237, 1610-1614]. Different sizes of mRNA found in liver (2.5 kilobases) and kidney (2 kilobases) suggest there may be tissue-specific processing of the primary transcript of this gene. Identification of human thyroid hormone receptor type alpha 2 indicates that two or more forms of thyroid hormone receptor exist in human tissues and may explain the normal variation in thyroid hormone responsiveness of various organs and the selective tissue abnormalities found in the thyroid hormone resistance syndromes.
G protein mRNA mapped in rat brain by in situ hybridization.
Authors: Largent B L , Jones D T , Reed R R , Pearson R C , Snyder S H
Published: May 2006

Guanine nucleotide-binding regulatory proteins (G proteins) mediate many receptor-coupled signal transduction events. We have localized in rat brain by in situ hybridization the mRNA for the G protein subunits--G alpha s, G alpha o, and G beta. Oligonucleotide probes were radiolabeled by a technique that resulted in a probe of defined specific activity and uniform length. mRNA species encoding G alpha s and G beta occur in high densities heterogeneously throughout the brain, especially in large neuronal cell bodies--e.g., hippocampal pyramidal cells, granule cells of the dentate gyrus, hypothalamic nuclei, and neurons of brainstem nuclei and the reticular formation. G alpha o mRNA has a more limited distribution and abundance, being detectable in the claustrum, endopiriform nucleus, habenula, hippocampal pyramidal cells, granule cells of the dentate gyrus, and cerebellar Purkinje cells.
Soluble interleukin 2 receptors are released from the cell surface of normal murine B lymphocytes stimulated with interleukin 5.
Authors: Loughnan M S , Sanderson C J , Nossal G J
Published: May 2006

Murine T and B lymphocytes can be induced to release soluble interleukin 2 receptors (IL2Rs). This receptor is believed to be a truncated form of the 55-kDa chain of the cell-membrane-associated receptor. It has been speculated that this receptor may play an important immunoregulatory role by binding to interleukin 2 (IL-2). We report here that interleukin 5 can induce normal murine B cells to release soluble IL2Rs. This extends our finding that interleukin 5 similarly can induce murine B cells to express functional cell-surface-associated IL2Rs. Two possible mechanisms of release of soluble IL2Rs have been suggested. Soluble IL2R could be synthesized as a secretory form of the receptor lacking the transmembrane domain or by cleavage of the extracellular domain of the cell-surface-associated IL2R at the cell surface. To investigate which mechanism was operative, we radioiodinated the cell surface of normal murine splenocytes that had been cultured for 1 day with Con A to stimulate the expression of cell-surface-associated IL2Rs and the release of soluble IL2Rs. Under the conditions used, radiolabeling of internal proteins was not apparent. Labeled cells were then recultured with Con A, conditioned medium was taken from replicate cultures at various times after radioiodination, and the specific radioactivity of released soluble IL2Rs was determined by ELISA and RIA. We demonstrate that the specific radioactivity and the kinetics of change of the specific radioactivity are consistent with the hypothesis that the soluble IL2Rs are derived from the cell-surface-associated IL2Rs rather than being released in a secretory form.
Rate, origin, and bidirectionality of Caulobacter chromosome replication as determined by pulsed-field gel electrophoresis.
Authors: Dingwall A , Shapiro L
Published: May 2006

Cell division in Caulobacter crescentus yields progeny cells that differ with respect to cell structure and developmental program. Chromosome replication initiates in the daughter stalked cell but is repressed in the daughter swarmer cell until later in the cell cycle. To study cell-type-specific DNA initiation, chromosome replication was directly analyzed by pulsed-field gel electrophoresis. Analysis of Dra I restriction fragments of DNA taken at various times from synchronized cell cultures labeled with 2'-deoxy[3H]guanosine has allowed us to determine the origin of DNA replication, the rate and direction of fork movement, and the order of gene replication. The first labeled Dra I fragment to appear contains the site of replication initiation. Based on the correlation of the physical and genetic maps derived by Ely and Gerardot [Ely, B. & Gerardot, C. J. (1988) Gene 68, 323-333], the origin was localized to a 305-kilobase fragment containing the rrnA gene. Furthermore, the sequential replication through unmapped Dra I fragments has enabled us to localize their positions on the genome. The order of appearance of labeled restriction fragments revealed that the chromosome replicates bidirectionally at a fork movement rate of 21 kilobases per minute.