bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple chromosomal rearrangements in a spontaneously arising t(6;7) rat immunocytoma juxtapose c-myc and immunoglobulin heavy chain sequences.
Authors: Pear W S , Ingvarsson S , Steffen D , Münke M , Francke U , Bazin H , Klein G , Sümegi J
Published: May 2006

Spontaneously arising immunocytomas in Lou/Wsl rats contain a consistent translocation between chromosomes 6 and 7. The c-myc gene has been localized to chromosome 7 and has been shown to be rearranged in the majority of the rat immunocytomas. We now report the cloning of the rearranged 11-kilobase EcoRI c-myc fragment from the IgE-secreting IR75 tumor. Sequence analysis revealed that the cytogenetically visible t(6;7) translocation must have involved several events in this tumor. One event has led to the juxtaposition of c-myc and the switch mu region, in a head-to-head orientation. The breakpoint is approximately 850 base pairs upstream from the proximal c-myc promoter on chromosome 7. This area is distinct from the more common mouse plasmacytoma- and Burkitt lymphoma-associated translocation breakpoints and also differs from the known murine retroviral insertion sites. A second rearrangement has led to the transposition of sequences upstream from the switch gamma 1 region to the c-myc-distant end of the switch mu region, tail-to-tail. This requires at least two events, including one inversion. In addition to showing that identical loci (c-myc, immunoglobulin) are juxtaposed via chromosomal translocations in three different tumors (Burkitt lymphoma, mouse plasmacytoma, and rat immunocytoma) in different species (human, mouse, and rat), the multiple rearrangements in IR75 and some other tumors emphasize the selective value of c-myc activation by an immunoglobulin locus in the tumorigenic process.
Polymorphisms in the human haptoglobin gene cluster: chromosomes with multiple haptoglobin-related (Hpr) genes.
Authors: Maeda N , McEvoy S M , Harris H F , Huisman T H , Smithies O
Published: May 2006

We have found polymorphisms for the number of tandemly arranged haptoglobin-related (Hpr) genes in the haptoglobin gene cluster of Blacks. Genomic mapping and nucleotide sequence analysis indicate that two copies of the Hpr gene first resulted from unequal but homologous crossing-over in a region 3' to the haptoglobin (Hp) and the haptoglobin-related genes. Subsequent increases in the number of Hpr loci have occurred in some chromosomes. Among 25 American Blacks studied (15 were unrelated), 2 related individuals have one extra copy of the Hpr gene and 5 unrelated individuals have more than two extra Hpr genes. None of 26 Whites and one Oriental studied have extra copies. In one of the Blacks, six tandemly arranged Hpr genes were demonstrated in one chromosome by pulsed field gradient electrophoresis. His other chromosome had one Hpr gene. The tandem Hpr genes were found in individuals with the haptoglobin genotypes Hp2/Hp2 (3 of 3 tested) and Hp2/Hp1 (4 of 11 tested), but none were found in the Hp1/Hp1 individuals (11 tested). Fibroblast cell cultures from two Hp2/Hp1 heterozygotes were fused to mouse cells to obtain cell lines retaining a human chromosome 16 on which the haptoglobin gene cluster is located. DNA analysis of the hybrid cells showed that in both individuals the tandemly arranged Hpr genes are linked to the Hp2 allele. These results suggest that the multiple copies are associated with the Hp2 gene.
Developmental aspects of proline transport in rat renal brush border membranes.
Authors: Medow M S , Roth K S , Goldmann D R , Ginkinger K , Hsu B Y , Segal S
Published: May 2006

Proline uptake by rat renal brush border membrane vesicles from animals 7 days of age and older has been examined to delineate developmental changes in membrane function that may underlie the physiological hyperprolinuria of young animals. Although the two proline transport systems normally present in adult membranes were found in membranes from young animals, the proline "overshoot" resulting from a sodium ion gradient is minimal and increases with age of the animal from which the membranes were isolated. This is associated with a severalfold faster entry of 22Na into vesicles of the 7-day-old animal compared to entry into membranes prepared from adult kidneys. The very rapid dissipation of the sodium gradient thus diminishing the driving force for transmembrane proline movement may explain the changes in proline overshoot observed in membranes from young animals. The altered sodium permeability is consistent with the fact that young animals have a generalized inability to reabsorb other amino acids whose transport is known to be sodium gradient stimulated.
Inhibition of heat shock protein synthesis by heat-inducible antisense RNA.
Authors: McGarry T J , Lindquist S
Published: May 2006

We show that antisense RNAs transcribed from genes that are stably integrated into the genome can be used to inhibit the expression of an endogenous cellular gene. Drosophila tissue culture cells were stably transformed with a gene encoding a heat-inducible RNA complementary to the message for hsp26, one of the small heat shock proteins. These cells produced much less hsp26 after heat shock than did untransformed cells. The inhibition was highly specific: expression of the closely related heat shock proteins hsp22, hsp23, and hsp28 was unaffected. By varying the copy number of the antisense gene, the degree of inhibition was varied over a broad range. Reducing the rate of hsp26 synthesis did not appear to affect the synthesis of any other protein during either heat shock or recovery.
Thioredoxin-catalyzed refolding of disulfide-containing proteins.
Authors: Pigiet V P , Schuster B J
Published: May 2006

Thioredoxin, a known catalyst for reducing protein disulfides, was shown to catalyze efficiently the refolding of pancreatic RNase either from the reduced, denatured form or from the scrambled form containing oxidized but incorrectly paired disulfides. Thioredoxin was 1000-fold more efficient on a molar basis than the model dithiol, dithiothreitol, in reactivating reduced, denatured RNase, suggesting that thioredoxin acts as an efficient catalyst for disulfide interchange. Starting with reduced, denatured RNase, enzyme activity was recovered quantitatively with a t1/2 of 30 hr with 100 microM thioredoxin compared to only a 10-20% recovery of activity in the control using air oxidation. Oxygen further stimulated the effectiveness of thioredoxin severalfold. Thioredoxin was most effective in reactivating inactive scrambled RNase, which contained mispaired disulfides, showing a t1/2 of 2 hr. Reduced thioredoxin was optimal for catalyzing disulfide interchange in scrambled RNase, whereas oxidized thioredoxin was required for reactivation of the reduced, denatured species. Optimal reactivation of scrambled RNase required a mixture of reduced and oxidized thioredoxin. Addition of reduced thioredoxin after initiating refolding of reduced denatured RNase with oxidized glutathione effected a rapid reactivation of RNase, suggesting a two-step model for protein refolding in which the monothiol catalyzes the rapid initial formation of protein disulfides and thioredoxin catalyzes the second step of disulfide interchange. Arguments are presented suggesting that thioredoxin may serve an in vivo role analogous to the protein disulfide-isomerase (EC 5.3.4.1).
Cloning and expression of Bradyrhizobium japonicum uptake hydrogenase structural genes in Escherichia coli.
Authors: Zuber M , Harker A R , Sultana M A , Evans H J
Published: May 2006

To identify the structural genes for the components of Bradyrhizobium japonicum uptake hydrogenase (Mr 60,000 and 30,000), we have expressed these genes in Escherichia coli and shown that the products cross-react with antibodies to the respective hydrogenase subunits. We constructed subclones of overlapping DNA fragments from an uptake hydrogenase-complementing cosmid, pHU52 [Lambert, G. R., Cantrell, M. A., Hanus, F. J., Russell, S. A., Haddad, K. R. & Evans, H. J. (1985) Proc. Natl. Acad. Sci. USA 82, 3232-3236], in pMZ 545, a plasmid expression vector. DNA fragments inserted into one or more of the four cloning sites downstream from the E. coli lac operon promoter (Plac) on pMZ 545 generate transcriptional, but not translational, fusions. Two subclones that directed the synthesis of Mr 60,000 and 30,000 proteins in E. coli "maxicells" were identified. The DNA inserts from these subclones were then inserted down-stream of the bacteriophage lambda PL promoter on a transcriptional fusion vector. When the PL promoter was activated in vivo by heat inactivation of the temperature sensitive cI repressor of lambda in an appropriate E. coli strain, the respective fragments expressed higher levels of Mr 60,000 and 30,000 proteins that could be detected in immunoblots. These data provide direct evidence for the presence of uptake hydrogenase structural genes on the uptake hydrogenase-complementing cosmid pHU52.
Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC.
Authors: Nixon B T , Ronson C W , Ausubel F M
Published: May 2006

We report that the ntrB and ntrC proteins of Bradyrhizobium sp. [Parasponia] strain RP501 share homology with other regulatory proteins. There is extensive conservation of C-terminal regions between products of RP501 ntrB; Klebsiella pneumoniae ntrB; Escherichia coli envZ, cpxA, and phoR; Agrobacterium tumefaciens virA; and, to a lesser extent, E. coli cheA. There is also extensive conservation of N-terminal regions between products of RP501 ntrC; K. pneumoniae ntrC; E. coli ompR, sfrA, phoB, cheY and cheB; Salmonella typhimurium cheB and cheY; Bacillus subtilis spoOA and spoOF; and A. tumefaciens virG. We propose that these regulatory genes comprise two-component regulatory systems that evolved from a common ancestral system that involved transduction of information about the status of the environment by one protein domain (the C-terminal regions conserved among ntrB, envZ, etc.) to a second one (the N-terminal region conserved among ntrC, ompR, etc.). The ntrC-set protein then acts upon a specific responding mechanism, typically as a transcriptional activator but also as an effector of the maturation of outer membrane proteins or as a modulator of the direction of flagella rotation.
Sodium-dependent regenerative responses in dendrites of axotomized motoneurons in the cat.
Authors: Sernagor E , Yarom Y , Werman R
Published: May 2006

Ten days after extradural axotomy, partial spikes are found in greater than 20% of cat L7 motoneurons, while 15-21 days after axotomy the incidence increases to 60%. These responses are produced in excitable (hot) spots in the dendrites by synaptic excitation. Intracellular injection of QX-314, a lidocaine derivative and effective blocker of Na+ channels from within neurons, results in elimination of partial spikes before blocking somadendritic spikes. The action of QX-314 does not depend on changes in passive membrane properties or on changes in synaptic properties. Injections of Cs+ and Cl- ions rule out any major role for calcium, potassium, or chloride currents in the production of partial spikes. The partial spikes represent an unusual Na+-dependent dendritic phenomenon induced by axotomy when carried out relatively near the soma. It is reasonable to postulate that the partial spikes result from a higher concentration of Na+ channels in the dendrites. This may be the consequence of a high rate of production of Na+-channel proteins that are intended for the cut end of the axon; alternatively, they may result from the reflection from the cut end of such proteins produced at either a normal or an increased rate. These aberrant channels are inserted into somatic and dendritic membranes in higher concentrations than normal and, as well as producing local dendritic regions of low safety factor responsible for the partial spikes, also produce somadendritic spikes of unusually fast rise time and lower than usual threshold, which are relatively resistant to QX-314.
Substance B: an endogenous brain factor that reverses presynaptic inhibition of acetylcholine release.
Authors: Pearce L B , Benishin C G , Cooper J R
Published: May 2006

Evidence is presented of the existence of a substance in brain (substance B) that reverses presynaptic inhibition of evoked acetylcholine (AcCho) release. Addition of guinea pig brain synaptosomal incubation medium or whole brain extracts to assays in which 1,1-dimethyl-4-phenylpiperazinium-evoked release of [3H]AcCho from guinea pig myenteric plexus synaptosomes reversed purinergic, muscarinic, and alpha 2-adrenergic agonist inhibition. However, the extracts did not increase basal or evoked release of AcCho. Results of chromatography on Bio-Gel P-2 of a concentrated whole brain extract suggest this factor has an Mr of 700. Substance B was resistant to boiling under acid or alkaline conditions as well as incubation with phospholipase C and various proteases; ashing, however, completely destroyed activity. This endogenous factor was also found to antagonize agonist-mediated inhibition of electrically evoked contractions of myenteric plexus-longitudinal muscle strips from the guinea pig ileum. The demonstration of substance B with its ability to reverse presynaptic inhibition of AcCho release reveals one mode of presynaptic modulation.
Isotope effect on electron transfer in reaction centers from
Rhodopseudomonas sphaeroides
Authors: Okamura M. Y. , Feher G.
Published: May 2006

Previous ENDOR studies on reaction centers from
Rhodopseudomonas sphaeroides
have shown the presence of two hydrogen-bonded protons associated with the primary, ubiquinone, acceptor Q
A
. These protons exchange with deuterons from solvent
2
H
2
O. The effect of this deuterium substitution on the charge-recombination kinetics (BChl)
2
+
Q
A
-
→ (BChl)
2
Q
A
has been studied with a sensitive kinetic difference technique. The electron-transfer rate was found to increase with deuterium exchange up to a maximum Δ
k/k
of 5.7 ± 0.3%. The change in rate was found to have an exchange time of 2 hr, which matched the disappearance of the ENDOR lines due to the exchangeable protons. These results indicate that these protons play a role in the vibronic coupling associated with electron transfer. A simple model for the isotope effect on electron transfer predicts a maximum rate increase of 20%, which is consistent with the experimental results.
Response of two pea hybrids to CO2 enrichment: a test of the energy overflow hypothesis for alternative respiration.
Authors: Musgrave M E , Strain B R , Siedow J N
Published: May 2006

Two pea (Pisum sativum L.) hybrids differing in the presence or absence of the cyanide-resistant (alternative) pathway of respiration were constructed by reciprocally crossing cv. Alaska and cv. Progress No. 9. The F1 hybrids were grown in greenhouses maintained at either 350 or 650 ppm CO2, and the growth, flowering, and dry matter accumulation were compared. The objective was to assess the significance of the alternative respiratory pathway to whole-plant carbon budgets and further to test the hypothesis that the alternative pathway is important in oxidizing excess carbohydrates such as might accumulate under conditions of CO2 enrichment. More carbohydrates were available in the F1 hybrid lacking the pathway, as evidenced by greater plant height, leaf area, specific leaf weight, and total dry matter compared with the reciprocal hybrid, especially at 650 ppm CO2. Specific leaf weight increased markedly under CO2 enrichment in the hybrid lacking the pathway, while it was the same at 350 and 650 ppm in the reciprocal cross. The hybrid lacking the alternative pathway also outperformed the reciprocal cross in terms of total dry matter and seed production. Increased branching with CO2 enrichment was observed in the hybrid lacking the pathway, while branching in the reciprocal cross was only slightly stimulated. These results suggest that alternative respiration consumes luxury carbohydrate and that respiration via this pathway may be considered energetically wasteful in terms of whole-plant carbon budgets.
Potentiation of growth factor activity by exogenous c-myc expression.
Authors: Sorrentino V , Drozdoff V , McKinney M D , Zeitz L , Fleissner E
Published: May 2006

The c-myc oncogene has been implicated in deregulation of cell growth in neoplastic cells and response to "competence-inducing" growth factors in normal cells. In the latter case, expression of c-myc has been shown to be associated with the transition from the G0 to the G1 phase of the cell cycle induced by platelet-derived growth factor (PDGF). In the work reported here, we have introduced the c-myc coding region, in a retroviral vector, into mouse and rat cells. We show that under conditions of anchorage-independent growth, constitutive c-myc expression increases the response of rodent cells to PDGF, as well as to other growth factors of both the competence-inducing and "progression" classes. These effects of the myc product are observed whether or not an exogenous ras gene has also been introduced into the same cells. Possible models for the influence of myc on growth responses are discussed.
Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells.
Authors: Bredberg A , Kraemer K H , Seidman M M
Published: May 2006

A shuttle vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in cultured skin cells from a patient with the skin-cancer-prone, DNA repair-deficient disease xeroderma pigmentosum and in repair-proficient cells. After replication in the human cells, progeny plasmids were purified. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of Escherichia coli carrying a suppressible amber mutation in the beta-galactosidase gene. Plasmid survival in the xeroderma pigmentosum cells was less than that of pZ189 harvested from repair-proficient human cells. The point-mutation frequency in the 150-base-pair tRNA marker gene increased up to 100-fold with ultraviolet dose. Sequence analysis of 150 mutant plasmids revealed that mutations were infrequent at potential thymine-thymine dimer sites. Ninety-three percent of the mutant plasmids from the xeroderma pigmentosum cells showed G X C----A X T transitions, compared to 73% in the normal cells (P less than 0.002). There were significantly fewer transversions (P less than 0.002) (especially G X C----T X A) and multiple base substitutions (P less than 0.00001) than when pZ189 was passaged in repair-proficient cells. The subset of mutational changes that are common to ultraviolet-treated plasmids propagated in both repair-proficient and xeroderma pigmentosum skin cells may be associated with the development of ultraviolet-induced skin cancer in humans.
Biological properties of a recombinant human immunoglobulin epsilon-chain fragment.
Authors: Ishizaka T , Helm B , Hakimi J , Niebyl J , Ishizaka K , Gould H
Published: May 2006

A recombinant human immunoglobulin epsilon-chain gene expression product (rFc epsilon) was compared with a human E myeloma protein in the affinity for epsilon-chain Fc fragment receptors (Fc epsilon R) on cultured human basophils. The association-dissociation kinetics of the rFc epsilon-Fc epsilon R interaction are indistinguishable from that of E myeloma protein, indicating that rFc epsilon and IgE have identical affinity for the receptors. The recombinant gene product sensitizes cultured basophils for anti-IgE-induced histamine release. A dose-response curve of histamine release indicates that the gene product is equally efficient in transducing the signal for degranulation as the natural IgE. Both the rFc epsilon and IgE lost the affinity for Fc epsilon R by heating at 56 degrees C. Upon renaturation by passage through a solution of 6 M guanidine hydrochloride, rFc epsilon recovered both the affinity for Fc epsilon R and the original CD spectra. On the other hand, renaturation of heat-denatured IgE largely restored optical activity above 250 nm but restored neither the affinity for Fc epsilon R nor the CD spectrum below 220 nm. The results suggest that either the amino acid sequence or the carbohydrate present in the myeloma protein, but not the rFc epsilon, may interfere with refolding of the receptor-binding structures.
Action-potential duration and the modulation of transmitter release from the sensory neurons of
Aplysia
in presynaptic facilitation and behavioral sensitization
Authors: Hochner Binyamin , Klein Marc , Schacher Samuel , Kandel Eric R.
Published: May 2006

Presynaptic facilitation of transmitter release from
Aplysia
sensory neurons is an important contributor to behavioral sensitization of the gill and siphon withdrawal reflex. The enhanced release is accompanied by reduction of the serotonin-sensitive S current in the sensory neurons and a consequent increase in duration of the presynaptic action potential (ranging from 10% to 30%). We find that changes of similar magnitude in the duration of depolarizing voltage-clamp steps in sensory neurons in intact abdominal ganglia yield increases in synaptic potentials of 45-120%. In dissociated cell culture, these changes lead to increases of 25-60% in the synaptic potential. Prolongation of presynaptic depolarization using voltage clamp or prolongation of the duration of the action potential by K
+
-channel blockers leads to prolongation of the time-to-peak of the synaptic potentials; similar changes in time-to-peak occur during presynaptic facilitation. The time-to-peak is not changed by homosynaptic depression or by changing the Ca
2+
concentration, procedures that alter release without changing the duration of the action potential. Preventing the spike from broadening by voltage clamping the presynaptic neuron substantially reduces or blocks the facilitation. These results suggest that broadening of the action potential during facilitation is a causal factor in the enhancement of transmitter release.
Molecular models for the putative dimer of sea lamprey hemoglobin.
Authors: Honzatko R B , Hendrickson W A
Published: May 2006

The known structures for the tetramers of mammalian and clam hemoglobins provide a point of departure for the modeling of putative dimers of lamprey hemoglobin. The association of subunits is dissimilar for the clam and mammalian tetramers; the superposition of the molecular model for lamprey methemoglobin onto the mammalian and clam tetramers gives five distinct dimers. After energy minimization of the interface regions of the five models, three models afford promising interactions between side chains. One model is analogous to the alpha 1 beta 2 pairing of subunits of mammalian hemoglobins. The other two models are similar to the interfaces between the E and F helices and between the A and B helices of clam hemoglobin. Although the model based on the alpha 1 beta 2 mode of association provides the best explanation of biochemical properties of lamprey hemoglobin, such as the Bohr effect and the dependency of dimer formation on pH, interfaces between the E and F and the A and B helices could be important in the aggregation of monomers of lamprey hemoglobin beyond the level of the dimer.
Polyadenylylation of an mRNA precursor occurs independently of transcription by RNA polymerase II in vivo.
Authors: Lewis E D , Manley J L
Published: May 2006

Most eukaryotic messenger RNAs are transcribed as precursor molecules that must be processed by capping, splicing, 3' cleavage, and polyadenylylation to yield mature mRNAs. An important, unresolved issue is whether any of these reactions are linked either to transcription by RNA polymerase II or to each other. To address one aspect of this question, we constructed a chimeric gene containing an RNA polymerase III promoter (the adenovirus VAI promoter) fused to the body and 3'-flanking sequences of a protein-coding gene (the herpesvirus tk gene). Here we show that this hybrid gene was transcribed from the RNA polymerase III promoter following transfection of human 293 cells and that the transcripts produced were stable and efficiently transported to the cytoplasm. Although a significant proportion of the transcripts were prematurely terminated at specific sites within the gene, a high percentage of the full-length RNA was accurately cleaved and polyadenylylated. These results demonstrate that cleavage and polyadenylylation of mRNA precursors are not obligatorily coupled to transcription by RNA polymerase II in vivo.
Stable repression of ribosomal protein L1 synthesis in Xenopus oocytes by microinjection of antisense RNA.
Authors: Wormington W M
Published: May 2006

The synthesis of an endogenous ribosomal protein, L1, is selectively and efficiently inhibited by microinjection of antisense L1 RNAs into Xenopus oocytes. Repression of L1 synthesis is achieved within 12 hr and is maintained for 48 hr. RNase-protection assays reveal the formation of RNA X RNA duplexes in vivo between the endogenous L1 mRNA and injected antisense transcripts. Partial-length antisense RNAs, complementary to only the 3'-terminal region of L1 mRNA, repress translation as effectively as a full-length antisense RNA, indicating that complementarity to the 5' region of L1 mRNA is not required for efficient inhibition. The use of antisense RNA to repress synthesis of an endogenous ribosomal protein provides a functional basis for determining mechanisms that integrate ribosomal protein synthesis with ribosome assembly during oogenesis.
Evolution of mitochondrial DNA in
Drosophila subobscura
Authors: Latorre Amparo , Moya Andrés , Ayala Francisco J.
Published: May 2006

The colonization of the New World by the Palearctic species
Drosophila subobscura
was first detected in 1978 in South America and around 1982 in western North America. The ensuing dramatic expansion of the species, in territory as well as numbers, provides an opportunity for studying evolution in a scale rarely possible. We have used 10 restriction endonucleases to analyze the mitochondrial DNA (mtDNA) of individuals from 23 widely dispersed localities. Only two mtDNA composite morphs have been detected in the Americas. None of the two morphs has been found in Africa, and only one in the Atlantic islands; but both are widespread in Europe, which provides no clue of the precise geographic origin of the colonizers. The amount of nucleotide-substitution polymorphism detected in
D. subobscura
is typical for animals, but it is greater in the Old than in the New World, presumably due to the recent colonization by a limited number of colonizers. Assuming standard evolutionary rates of mtDNA base substitution, the mtDNA morphs found in
D. subobscura
can be traced to a single one that existed no less than one million years ago. We argue against the inference that the
D. subobscura
flies now living descend from only one or a few females that lived at that time. This type of inference, which we call the “Mother Eve hypothesis,” has been made to conclude that the human population went through a severe constriction about 200,000 years ago, so that all living humans descend from only one or a few women who lived at that time. The Mother Eve hypothesis is fallacious.
Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase.
Authors: Smith D B , Davern K M , Board P G , Tiu W U , Garcia E G , Mitchell G F
Published: May 2006

Mice of the inbred strain 129/J bred at this Institute (WEHI 129/J) are relatively resistant to chronic infection with the parasitic helminth Schistosoma japonicum. In contrast to more permissive mouse strains such as BALB/c, the WEHI 129/J mice are high responders to a Mr 26,000 adult worm antigen designated Sj26. Cloned cDNAs corresponding to Sj26 have been identified in a S. japonicum phage lambda gt11 amp3 expression library, and their nucleotide sequences have been deduced. The predicted amino acid sequence of the antigen specified by these cDNAs shows striking homology with class mu isozymes of mammalian glutathione S-transferases (RX:glutathione R-transferase, EC 2.5.1.18). Extracts of adult worms contain glutathione S-transferase activity, and affinity chromatography of enzyme activity on glutathione columns leads to the purification of a Mr 26,000 molecule that comigrates with Sj26. Although vaccination studies in mice with a beta-galactosidase-Sj26 fusion protein from Escherichia coli are encouraging, more immunogenic preparations of the antigen are likely to be required to establish the utility of Sj26 as a model vaccine.
Extensive variation and heteroplasmy in size of mitochondrial DNA among geographic populations of
Drosophila melanogaster
Authors: Hale Lawrence R. , Singh Rama S.
Published: May 2006

Size variation and heteroplasmy in mitochondrial DNA (mtDNA) are relatively common in natural populations of
Drosophila melanogaster
. Of 92 isofemale lines of flies obtained from various geographic regions throughout the world, 75 lines were homoplasmic and showed a total of 12 different mtDNA size classes. The remaining 17 lines were heteroplasmic, each line carrying two different mtDNAs, and, in all but one case, the mtDNAs in these heteroplasmic lines differed in size; a total of nine size classes was represented among them. In cases where one type was predominant within an individual, it was usually the smaller mtDNA. This finding parallels what was observed in homoplasmic lines, in that the smaller mtDNAs were much more common than the larger variants in most populations. The data suggest a high rate of mutational occurrence of mtDNA size variants and some natural selection against them.
Nuclease resistance and the enrichment of native nuclear acceptor sites for the avian oviduct progesterone receptor.
Authors: Hora J , Horton M J , Toft D O , Spelsberg T C
Published: May 2006

High-affinity nucleoprotein acceptor sites for the avian oviduct progesterone receptor (PR) have been enriched by a combination of nuclease digestion and centrifugation. These enriched binding elements exhibited markedly enhanced PR binding on a per mass DNA basis compared to chromatin (20- to 25-fold) or dehistonized chromatin (4- to 5-fold). Electrophoretic analysis of the nuclease-resistant DNA showed that there is a set of DNA fragments of 100-150 base pairs that are protected from digestion. Excessive digestion resulted in smaller DNA fragments and a loss of PR binding activity. The PR binding was saturable using a crude receptor preparation and displayed a competition with the same receptor preparation that was labeled with nonradioactive progesterone. The enhanced binding was also demonstrable using highly purified receptor preparations that exhibit two classes of binding sites both of which are of high affinity and saturable as assessed by Scatchard analyses. These two high-affinity classes of binding sites are shown to be competed by unlabeled purified PR. The nuclease resistance of these nucleoprotein acceptor sites from chromatin is a property similar to the nuclear matrix binding sites suggesting a relationship between these two classes of nuclear acceptor sites.
Identification of the protein encoded by the human diffuse B-cell lymphoma (dbl) oncogene.
Authors: Srivastava S K , Wheelock R H , Aaronson S A , Eva A
Published: May 2006

The dbl oncogene was initially isolated from a human diffuse B-cell lymphoma. Antisera from mice bearing tumors induced by this oncogene specifically detected a protein of about 66 kDa (p66) in dbl transformants. dbl cDNA-selected poly(A)+ RNA isolated from a transfectant clone expressing p66 directed the in vitro synthesis of this protein, establishing that it is encoded by dbl. Subcellular localization studies revealed that p66 is a cytoplasmic protein distributed between cytosol and crude membrane fractions. Moreover, p66 was shown to be a phosphoprotein, with phosphorylation specific to serine residues. Our characterization of the dbl-encoded protein appears to distinguish this transforming gene product from those of other known oncogenes.
Preferential DNA repair of an active gene in human cells.
Authors: Mellon I , Bohr V A , Smith C A , Hanawalt P C
Published: May 2006

Removal of pyrimidine dimers was measured in defined sequences in human cells amplified for the dihydrofolate reductase (DHFR) gene. We quantitated repair in specific restriction fragments by using the dimer-specific bacteriophage T4 endonuclease V and analysis by Southern blotting. Within 4 hr after 5- or 10-J/m2 UV irradiation, more than 60% of the dimers had been removed from a 20-kilobase fragment that lies entirely within the transcription unit of the DHFR gene and from a 25-kilobase fragment located in the 5' flanking region of the gene. Repair in the overall genome was measured by analyzing cellular DNA treated with T4 endonuclease V in alkaline sucrose gradients. Sixty-nine percent of the dimers were removed from the genome overall within 24 hr after irradiation, but only 25% were removed within 4 hr and 38% were removed within 8 hr. These results demonstrate a strong preferential rate of removal of dimers from the 50-kilobase region that includes the transcriptionally active DHFR gene compared to that in total cellular DNA. We confirmed that DHFR-containing DNA is repaired more rapidly than bulk DNA by using an approach that provides a direct comparison between repair in specific sequences and repair in total cellular DNA. We also show that the DHFR-containing sequences are repaired more rapidly than the nontranscribed repetitive alpha DNA sequences. Our finding of preferential early repair in a transcriptionally active region in overall repair-proficient cells suggests that selective dimer removal from active sequences may be a general characteristic of mammalian DNA repair.
Post-separation detection of nucleic acids and proteins by neutron activation.
Authors: Snapka R M , Kwok K , Bernard J A , Harling O K , Varshavsky A
Published: May 2006

We describe approaches to neutron activation analysis and their application to post-separation autoradiographic detection of biological compounds. Specifically, we have extended the use of a "direct-labeling" method to the post-separation detection of DNA after gel electrophoresis and to the detection of nucleotides separated by TLC. In addition, we describe a more generally applicable "indirect-labeling" method in which separated compounds of interest are selectively bound to ligands containing highly neutron-activatable elements, such as manganese (55Mn), europium (151Eu), or dysprosium (164Dy), and then irradiated with thermal neutrons. This method is illustrated with nucleotides separated by TLC and with proteins separated by polyacrylamide gel electrophoresis. In contrast to the direct-labeling approach, the indirect-labeling method can be adapted to detect any class of substances for which a highly neutron-activatable, selectively binding ligand is available. The theoretically achievable sensitivity of the indirect-labeling method is in the attomole (10(-18) mol) range.
Resinless section electron microscopy of HeLa cell mitotic architecture.
Authors: Wagner B , Krochmalnic G , Penman S
Published: May 2006

The use of resinless sections extends embedment-free electron microscopy to the cytoskeleton of thick specimens. Here we examine HeLa cells rounded at mitosis. Extraction of mitotic HeLa cells with Triton X-100 removes lipids and soluble proteins, leaving the cytoskeletal framework and spindle apparatus. After fixation, the samples are embedded and sectioned, and the temporary embedding resin is removed for direct visualization in the electron microscope. The micrographs show that the cytoskeletal framework, chromosomes, spindle, and centrioles form an interconnected entity. The pericentriolar region, indistinct in conventional micrographs, appears composed of distinct fibers interconnecting the spindle microtubules and centriole. The resinless sections also reveal characteristic lacunae at late anaphase/early telophase. These probably result from reformation of the interphase cytoskeleton lagging reassembly of the nucleus.
Isolation of CD1 genes: a family of major histocompatibility complex-related differentiation antigens.
Authors: Martin L H , Calabi F , Milstein C
Published: May 2006

CD1 differentiation antigens are defined by a group of monoclonal antibodies that characterize immature human thymocytes. A cloned cDNA has been used to identify CD1 genes in a human genomic library. Five CD1 genes have been isolated, and Southern blot analysis suggests that these represent all the cross-hybridizing human CD1 genes. They share a highly conserved exon, which is homologous to the beta 2-microglobulin-binding domain (alpha 3) of major histocompatibility complex (MHC) class I antigens. In this domain, amino acid sequences are 71-88% homologous. However, the homology between CD1 and MHC class I alpha 3 domains is only 21%. This is the same degree of homology as between either of them and the class II beta 2 domain, which does not bind beta 2-microglobulin. The evolutionary implications of these results are discussed.
Presence of the peroxisomal 22-kDa integral membrane protein in the liver of a person lacking recognizable peroxisomes (Zellweger syndrome).
Authors: Lazarow P B , Fujiki Y , Small G M , Watkins P , Moser H
Published: May 2006

Peroxisomes have not been detected in liver and kidney of patients with Zellweger syndrome. Some peroxisome proteins are missing; others are present in normal amounts but are located in the cytosol. We have prepared an antiserum against the 22-kDa integral membrane protein characteristic of rat liver peroxisomes. The antiserum crossreacts with the human liver counterpart, which likewise has a mass of 22 kDa. By immunoblot analysis, we demonstrate that the 22-kDa protein is present in normal amount in Zellweger liver and is integral to a membrane. The result suggests that peroxisome membranes are assembled in Zellweger syndrome but may be defective for the import of matrix proteins. As a result, newly synthesized proteins are left in the cytosol, where some persist and others are degraded. Lacking their usual content, such aberrant peroxisomal membranes would be unrecognizable morphologically. Immunoblot analyses also showed that the peroxisomal hydratase-dehydrogenase is deficient in Zellweger kidney as well as liver, but catalase is present in both organs.
Two-dimensional modeling of visual receptive fields using Gaussian subunits.
Authors: Soodak R E
Published: May 2006

Retinal ganglion cell receptive fields have been successfully described using the difference of Gaussians model introduced by Rodieck. As the basic elements of retinal receptive fields are well described by the Gaussian function, it is natural to model receptive fields beyond this level as a convergence of Gaussian subunits. In this paper the full two-dimensional solution to the problem of calculating the response to drifting gratings of a model receptive field composed of Gaussian subunits is presented. The subunits are not required to be radially symmetric, any number is allowed, with any temporal phase delays; and responses are predicted to gratings of any spatial frequency at any orientation. This solution will greatly extend the range of receptive fields that can be modeled as a convergence of Gaussian subunits, including those with orientational and directional selectivities.
Jimpy mutant mouse: a 74-base deletion in the mRNA for myelin proteolipid protein and evidence for a primary defect in RNA splicing.
Authors: Nave K A , Lai C , Bloom F E , Milner R J
Published: May 2006

The mouse mutant jimpy carries an X chromosome-linked recessive gene defect that affects the formation of myelin in the central nervous system. To understand the molecular basis of the jimpy mutation, we have examined the expression of mRNAs encoding myelin proteolipid protein (PLP). PLP mRNAs were detectable in jimpy brain RNA at 21 days after birth but were severely reduced in abundance compared to wild-type littermates. Nucleotide sequence analysis of cDNA clones for PLP mRNA, isolated from a cDNA library of jimpy brain poly(A)+ RNA, revealed that the PLP mRNA expressed in jimpy contained a deletion of 74 nucleotides with respect to the wild-type sequence. This deletion causes a frameshift in the open reading frame resulting in an altered carboxyl terminus for jimpy PLP. Probes specific for the deleted sequence, however, hybridize with equal efficiency to genomic DNA from jimpy and wild-type littermates, suggesting that the defect in the jimpy PLP mRNA is generated by aberrant RNA processing rather than by deletion of genomic sequences. We conclude that a mutation in the gene for PLP that leads to an incorrectly spliced RNA transcript is the primary defect of this genetic disorder.
Expression of the nodulation gene
nodA
in
Rhizobium meliloti
and localization of the gene product in the cytosol
Authors: Schmidt Jürgen , John Michael , Wieneke Ursula , Krüssmann Heinz-Dieter , Schell Jeff
Published: May 2006

The
nodA
gene of
Rhizobium meliloti
encodes a 21.8-kDa protein, which is conserved in several
Rhizobium
species. We overproduced the nodA protein as a fusion product with a portion of the λ
c
I repressor in
Escherichia coli
. This fusion protein was purified from inclusion bodies by gel and hydroxyapatite chromatography in the presence of NaDodSO
4
. Monospecific polyclonal antibodies against the hybrid protein were used to detect the nodA protein in the cytosol of
E. coli
and
R. meliloti
by immunoblotting. In contrast to experiments with antibodies against the
R. meliloti
nodC membrane protein, the alfalfa-
R. meliloti
nodulation was not affected by the addition of anti-nodA antibodies to medium and inoculum. This suggests that the nodA protein is located within the cell and is therefore not accessible to antibodies. The expression of the
nodA
gene is induced in
R. meliloti
by various compounds present in the exudate of leguminous plants, particularly by the flavone luteolin. We show that the plant hormone trigonelline also has some inducing activity. The nodC protein was further localized in the membrane fraction of
R. meliloti
. Our experiments demonstrate that the nodC transmembrane protein is not necessary for the uptake of the compounds inducing the synthesis of the nodA protein. The nodA and the nodC proteins were also detected in mature nodules. During nodule development, the nodC protein may be processed to a 34-kDa protein.
Rapid plasmid library screening using RecA-coated biotinylated probes.
Authors: Rigas B , Welcher A A , Ward D C , Weissman S M
Published: May 2006

A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.
Transvenous coronary angiography in humans using synchrotron radiation.
Authors: Rubenstein E , Hofstadter R , Zeman H D , Thompson A C , Otis J N , Brown G S , Giacomini J C , Gordon H J , Kernoff R S , Harrison D C
Published: May 2006

The risks and costs of the present method of visualizing the coronary arteries have limited the use of coronary angiography in long-term serial studies needed to establish the natural history of coronary atherosclerosis and its response to interventions. A less invasive method, in which the contrast agent is administered intravenously, has been developed using synchrotron radiation as the illuminating source. The present report describes the initial results in human subjects. The findings indicate that transvenous coronary angiograms can be acquired in this manner. Further refinements in the x-ray imaging system are expected to result in increased x-ray fluence and improved image quality.
Increased in vitro phosphorylation of a Mr 60,000 protein in brain from patients with Alzheimer disease.
Authors: Saitoh T , Dobkins K R
Published: May 2006

We have established in vitro conditions under which we can reliably measure kinase activity in normal postmortem human brain. Using these conditions, we detected in the brains of patients with Alzheimer disease a 2-fold increase in the level of Mr 60,000 protein phosphorylation compared to age-matched controls. The Mr 60,000 protein phosphorylation was found exclusively in the cytosol fraction. No differences were detected between phosphoproteins in 100,000 X g pellet fractions from brains of Alzheimer disease patients and from age-matched controls. Postmortem time up to 17 hr does not seem to affect the phosphorylation level of the Mr 60,000 protein. Younger Alzheimer disease patients had more prominent changes in the elevation of the Mr 60,000 protein phosphorylation level than older patients, although in the control patient, age did not affect the phosphorylation level of the Mr 60,000 protein. We conclude that in the brain cytosol of Alzheimer disease there may be an abnormality in either the degree of Mr 60,000 protein phosphorylation or in the Mr 60,000 protein concentration.
Morphine and codeine from mammalian brain.
Authors: Weitz C J , Lowney L I , Faull K F , Feistner G , Goldstein A
Published: May 2006

Recently, we described the presence of six immunoreactive (ir) morphinans in bovine adrenal and hypothalamus and identified one as morphine [Goldstein, A., Barrett, R. W., James, I. F., Lowney, L. I., Weitz, C. J., Knipmeyer, L. L. & Rapaport, H. (1985) Proc. Natl. Acad. Sci. USA 82, 5203-5207]. We now report that ir morphinans corresponding to the previously reported peak 1 (morphine), peak 4, and peak 5 are consistently present in extracts of bovine hypothalamus and variably present in extracts of bovine adrenal and rat brain. We no longer detect the previously reported peaks 2, 3, or 6, and we have established that they were contamination artifacts. Peak 1 is coeluted with morphine in two distinct reversed-phase HPLC systems, as is peak 4 with codeine. We have purified peak 1 and peak 4 compounds from bovine hypothalamus and determined their identities by gas chromatography/mass spectrometry (GC/MS): peak 1 is confirmed to be morphine and peak 4 is codeine.
Dissipative structures in a two-cell system: Numerical and experimental approaches
Authors: Breton J. , Thomas D. , Hervagault J. F.
Published: May 2006

It has been shown that the coupling between the photoreduction of the oxidized form of dichloroindophenol (an artificial electron acceptor) by thylakoids and the incident light intensity can lead to the appearance of multiple steady states when the system is operated under open conditions. In the present work, a numerical study and experimental evidence are presented on the occurrence of dissipative structures in an arrangement of two continuously stirred tank reactors with mutual mass exchange of dichloroindophenol through an inert membrane. The stable spatial structures are generated by the creation of transient internal and external asymmetries. A nontrivial hysteresis effect between symmetric and asymmetric stable steady states has been observed.