bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis
Authors: Ramachandran Anup , Moellering Douglas R. , Ceaser Erin , Shiva Sruti , Xu Jun , Darley-Usmar Victor
Published: July 2002

Mutations in mitochondrial DNA, affecting the activity of respiratory complexes, have been implicated in many chronic degenerative diseases. Mitochondrial proteins coded for by both the mitochondrial and nuclear genes are known to have important signaling roles in apoptosis. However, the impact of the inhibition of mitochondrial protein synthesis on apoptosis is largely unknown. This inhibition is particularly important in NO-dependent cytotoxicity, which is believed to have a significant mitochondrial component and depend on other factors such as glycolysis. In this study we have examined whether the inhibition of mitochondrial protein synthesis by chloramphenicol increases the susceptibility of endothelial cells to undergo NO-dependent apoptosis in glucose-free media. Bovine aortic endothelial cells were treated with chloramphenicol, which resulted in a decreased ratio of mitochondrial complex IV to cytochromecand increased oxidant production in the cell. Inhibition of mitochondrial protein synthesis was associated with a greater susceptibility of the cells to apoptosis induced by NO in glucose-free medium.
An
in vitro
assay reveals essential protein components for the “catch” state of invertebrate smooth muscle
Authors: Yamada Akira , Yoshio Maki , Kojima Hiroaki , Oiwa Kazuhiro
Published: July 2002

“Catch,” a state where some invertebrate muscles sustain high
tension over long periods of time with little energy expenditure (low
ATP hydrolysis rate) is similar to the “latch” state of
vertebrate smooth muscles. Its induction and release involve
Ca
2+
-dependent phosphatase and cAMP-dependent protein
kinase, respectively. Molecular mechanisms for catch remain obscure.
Here, we describe a quantitative microscopic
in vitro
assay reconstituting the catch state with proteins isolated from catch
muscles. Thick filaments attached to glass coverslips and pretreated
with ≈10
−4
M free Ca
2+
and soluble muscle
proteins bound fluorescently labeled native thin filaments tightly in
catch at ≈10
−8
M free Ca
2+
in the presence
of MgATP. At ≈10
−4
M free Ca
2+
, the thin
filaments moved at ≈4 μm/s. Addition of cAMP and cAMP-dependent
protein kinase at ≈10
−8
M free Ca
2+
caused
their release. Rabbit skeletal muscle F-actin filaments completely
reproduced the results obtained with native thin filaments. Binding
forces >500 pN/μm between thick and F-actin filaments were
measured by glass microneedles, and were sufficient to explain catch
tension
in vivo
. Synthetic filaments of purified myosin
and twitchin bound F-actin in catch, showing that other components of
native thick filaments such as paramyosin and catchin are not
essential. The binding between synthetic thick filaments and F-actin
filaments depended on phosphorylation of twitchin but not of myosin.
Cosedimentation experiments showed that twitchin did not bind directly
to F-actin in catch. These results show that catch is a direct
actomyosin interaction regulated by twitchin phosphorylation.
Evidence that replication fork components catalyze establishment of cohesion between sister chromatids
Authors: Carson Dena R. , Christman Michael F.
Published: July 2002

Accurate chromosome segregation requires that replicated sister chromatids are held together until anaphase, when their “cohesion” is dissolved, and they are pulled to opposite spindle poles by microtubules. Establishment of new cohesion between sister chromatids in the next cell cycle is coincident with replication fork passage. Emerging evidence suggests that this temporal coupling is not just a coincident timing of independent events, but rather that the establishment of cohesion is likely to involve the active participation of replication-related activities. These include PCNA, a processivity clamp for some DNA polymerases, Trf4/Pol σ (formerly Trf4/Polκ), a novel and essential DNA polymerase, and a modified Replication Factor C clamp–loader complex. Here we describe recent advances in how cohesion establishment is linked to replication, highlight important unanswered questions in this new field, and describe a “polymerase switch” model for how cohesion establishment is coupled to replication fork progression. Building the bridges between newly synthesized sister chromatids appears to be a fundamental but previously unrecognized function of the eukaryotic replication machinery.
The subunit structure and catalytic mechanism of the
Bacillus subtilis
DNA repair enzyme spore photoproduct lyase
Authors: Rebeil Roberto , Nicholson Wayne L.
Published: July 2002

The major DNA photoproduct of dormant, UV-irradiated
Bacillus subtilis
spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)]. During spore germination, SP is reversed to two intact thymines
in situ
by the DNA repair enzyme SP lyase, an S-adenosylmethionine (S-AdoMet)-dependent iron-sulfur ([Fe-S]) protein encoded by the
splB
gene. In the present work, cross-linking, SDS/PAGE, and size exclusion chromatography revealed that SplB protein dimerized when incubated with iron and sulfide under anaerobic reducing conditions. SplB isolated under aerobic conditions generated an EPR spectrum consistent with that of a partially degraded [3Fe-4S] center, and reduction of SplB with dithionite shifted the spectrum to that of a [4Fe-4S] center. Addition of S-AdoMet to SplB converted some of the [4Fe-4S] centers to an EPR-silent form consistent with electron donation to S-AdoMet. HPLC and electrospray ionization MS analyses showed that SP lyase cleaved S-AdoMet to generate 5′-deoxyadenosine. The results indicate that (
i
) SP lyase is a homodimer of SplB; (
ii
) dimer formation is coordinated by a [4Fe-4S] center; and (
iii
) the reduced [4Fe-4S] center is capable of donating electrons to S-AdoMet to generate a 5′-adenosyl radical that is then used for the
in situ
reversal of SP. Thus, SP lyase belongs to the “radical SAM” superfamily of enzymes that use [Fe-S] centers and S-AdoMet to generate adenosyl radicals to effect catalysis. SP lyase is unique in being the first and only DNA repair enzyme known to function via this novel enzymatic mechanism.
Postsymbiotic plasmid acquisition and evolution of the
repA1
-replicon in
Buchnera aphidicola
Authors: Van Ham Roeland C. H. J. , González-Candelas Fernando , Silva Francisco J. , Sabater Beatriz , Moya Andrés , Latorre Amparo
Published: July 2002

Buchnera aphidicola
is an obligate, strictly
vertically transmitted, bacterial symbiont of aphids. It supplies its
host with essential amino acids, nutrients required by aphids but
deficient in their diet of plant phloem sap. Several lineages of
Buchnera
show adaptation to their nutritional role in
the form of plasmid-mediated amplification of key-genes involved in the
biosynthesis of tryptophan (
trpEG
) and leucine
(
leuABCD
). Phylogenetic analyses of these
plasmid-encoded functions have thus far suggested the absence of
horizontal plasmid exchange among lineages of
Buchnera
.
Here, we describe three new
Buchnera
plasmids, obtained
from species of the aphid host families Lachnidae and Pemphigidae. All
three plasmids belong to the
repA1
family of
Buchnera
plasmids, which is characterized by the
presence of a
repA1-
replicon responsible for replication
initiation. A comprehensive analysis of this family of plasmids
unexpectedly revealed significantly incongruent phylogenies for
different plasmid and chromosomally encoded loci. We infer from these
incongruencies a case of horizontal plasmid transfer in
Buchnera
. This process may have been mediated by
secondary endosymbionts, which occasionally undergo horizontal
transmission in aphids.
Molecular genetics and the evolution of ultraviolet vision in vertebrates
Authors: Shi Yongsheng , Radlwimmer F. Bernhard , Yokoyama Shozo
Published: July 2002

Despite the biological importance of UV vision, its molecular bases
are not well understood. Here, we present evidence that UV vision in
vertebrates is determined by eight specific amino acids in the UV
pigments. Amino acid sequence analyses show that contemporary UV
pigments inherited their UV sensitivities from the vertebrate ancestor
by retaining most of these eight amino acids. In the avian lineage, the
ancestral pigment lost UV sensitivity, but some descendants regained it
by one amino acid change. Our results also strongly support the
hypothesis that UV pigments have an unprotonated Schiff base-linked
chromophore.
Compared with saturated fatty acids, dietary monounsaturated fatty acids and carbohydrates increase atherosclerosis and VLDL cholesterol levels in LDL receptor-deficient, but not apolipoprotein E-deficient, mice
Authors: Merkel Martin , Velez-Carrasco Wanda , Hudgins Lisa C. , Breslow Jan L.
Published: July 2002

Heart-healthy dietary recommendations include decreasing the intake of saturated fatty acids (SFA). However, the relative benefit of replacing SFA with monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), or carbohydrates (CARB) is still being debated. We have used two mouse models of atherosclerosis, low density lipoprotein receptor-deficient (LDLRKO) and apolipoprotein E-deficient (apoEKO) mice to measure the effects of four isocaloric diets enriched with either SFA, MUFA, PUFA, or CARB on atherosclerotic lesion area and lipoprotein levels. In LDLRKO mice, compared with the SFA diet, the MUFA and CARB diets significantly increased atherosclerosis in both sexes, but the PUFA diet had no effect. The MUFA and CARB diets also increased very low density lipoprotein-cholesterol (VLDL-C) and LDL-cholesterol (LDL-C) in males and VLDL-C levels in females. Analysis of data from LDLRKO mice on all diets showed that atherosclerotic lesion area correlated positively with VLDL-C levels (males:
r
= 0.47,
P
< 0.005; females:
r
= 0.52,
P
< 0.001). In contrast, in apoEKO mice there were no significant dietary effects on atherosclerosis in either sex. Compared with the SFA diet, the CARB diet significantly decreased VLDL-C in males and the MUFA, PUFA, and CARB diets decreased VLDL-C and the CARB diet decreased LDL-C in females. In summary, in LDLRKO mice the replacement of dietary SFA by either MUFA or CARB causes a proportionate increase in both atherosclerotic lesion area and VLDL-C. There were no significant dietary effects on atherosclerotic lesion area in apoEKO mice. These results are surprising and suggest that, depending on the underlying genotype, dietary MUFA and CARB can actually increase atherosclerosis susceptibility, probably by raising VLDL-C levels through a non-LDL receptor, apoE-dependent pathway.
Top-down mass spectrometry of a 29-kDa protein for characterization of any posttranslational modification to within one residue
Authors: Sze Siu Kwan , Ge Ying , Oh HanBin , McLafferty Fred W.
Published: July 2002

A mass difference between the measured molecular weight of a protein and that of its DNA-predicted sequence indicates sequence errors and/or posttranslational modifications. In the top-down mass spectrometry approach, the measured molecular ion is dissociated, and these fragment masses are matched against those predicted from the protein sequence to restrict the locations of the errors/modifications. The proportion of the ion's interresidue bonds that are cleaved determines the specificity of such locations; previously, ubiquitin (76 residues) was the largest for which all such bonds were dissociated. Now, cleavages are achieved for carbonic anhydrase at 250 of the 258 interresidue locations. Cleavages of three spectra would define posttranslational modifications at 235 residues to within one residue. For 24 of the 34 possible phosphorylation sites, the cleavages of one spectrum would delineate exactly all −PO
3
H substitutions. This result has been achieved with electron-capture dissociation by minimizing the further cleavage of primary product ions and by denaturing the tertiary noncovalent bonding of the molecular ions under a variety of conditions.
neurogenin2
elicits the genesis of retinal neurons from cultures of nonneural cells
Authors: Yan Run-Tao , Ma Wen-Xin , Wang Shu-Zhen
Published: July 2002

neurogenin2
(
ngn2
) encodes a basic
helix–loop–helix transcription factor and plays an important
role in neurogenesis from migratory neural crest cells. Its role in
retinal development is poorly understood. We observed that in the
developing chick retina,
ngn2
was expressed in a
subpopulation of proliferating progenitor cells. Ectopic expression of
ngn2
in nonneural, retinal pigment epithelial cell
culture triggered
de novo
generation of cells
that expressed neural-specific markers and exhibited neuronal
morphologies. Further molecular and morphological analyses showed that
the main products of the induced neurogenesis were cells resembling
young photoreceptor cells and cells resembling retinal ganglion cells.
The generation of multiple cell types suggests that
ngn2
induces various retinal pathways. Thus, unlike in the peripheral
nervous system where
ngn2
specifies one type of sensory
neuron,
ngn2
in the retina is likely involved in a
common step leading to different cellular pathways. Our finding that
ngn2
can instruct nonneural retinal pigment epithelial
cells to differentiate toward retinal neurons demonstrates one possible
way to induce
de novo
retinal neurogenesis.
A Schistosoma mansoni fatty acid-binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine.
Authors: Tendler M , Brito C A , Vilar M M , Serra-Freire N , Diogo C M , Almeida M S , Delbem A C , Da Silva J F , Savino W , Garratt R C , Katz N , Simpson A S
Published: July 2002

Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel beta-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSm14, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively.
NMR studies of muscle glycogen synthesis in insulin-resistant offspring of parents with non-insulin-dependent diabetes mellitus immediately after glycogen-depleting exercise.
Authors: Price T B , Perseghin G , Duleba A , Chen W , Chase J , Rothman D L , Shulman R G , Shulman G I
Published: July 2002

To examine the impact of insulin resistance on the insulin-dependent and insulin-independent portions of muscle glycogen synthesis during recovery from exercise, we studied eight young, lean, normoglycemic insulin-resistant (IR) offspring of individuals with non-insulin-dependent diabetes mellitus and eight age-weight matched control (CON) subjects after plantar flexion exercise that lowered muscle glycogen to approximately 25% of resting concentration. After approximately 20 min of exercise, intramuscular glucose 6-phosphate and glycogen were simultaneously monitored with 31P and 13C NMR spectroscopies. The postexercise rate of glycogen resynthesis was nonlinear. Glycogen synthesis rates during the initial insulin independent portion (0-1 hr of recovery) were similar in the two groups (IR, 15.5 +/- 1.3 mM/hr and CON, 15.8 +/- 1.7 mM/hr); however, over the next 4 hr, insulin-dependent glycogen synthesis was significantly reduced in the IR group [IR, 0.1 +/- 0.5 mM/hr and CON, 2.9 +/- 0.2 mM/hr; (P < or = 0.001)]. After exercise there was an initial rise in glucose 6-phosphate concentrations that returned to baseline after the first hour of recovery in both groups. In summary, we found that following muscle glycogen-depleting exercise, IR offspring of parents with non-insulin-dependent diabetes mellitus had (i) normal rates of muscle glycogen synthesis during the insulin-independent phase of recovery from exercise and (ii) severely diminished rates of muscle glycogen synthesis during the subsequent recovery period (2-5 hr), which has previously been shown to be insulin-dependent in normal CON subjects. These data provide evidence that exercise and insulin stimulate muscle glycogen synthesis in humans by different mechanisms and that in the IR subjects the early response to stimulation by exercise is normal.
A new isoleucine substitution of Val-20 in transthyretin tetramers selectively impairs dimer-dimer contacts and causes systemic amyloidosis.
Authors: Jenne D E , Denzel K , Blätzinger P , Winter P , Obermaier B , Linke R P , Altland K
Published: July 2002

The most frequent form of inherited amyloidoses is associated with mutations in the transthyretin (TTR) gene coding for 127-amino acid residues of four identical, noncovalently linked subunits that form a pair of dimers in the plasma protein complex. Amyloid fibrils containing the variant and to a lesser extent the wild-type form of the TTR molecule are deposited in various organs, including peripheral nerves and the myocardium, with polyneuropathy and cardiomyopathy as major clinical manifestations. So far, more than 40 distinct amino acid substitutions distributed throughout the TTR sequence over 30 positions have been found to be correlated with an increased amyloidogenicity of TTR. Most of these amyloidogenic amino acid substitutions are suspected to alter the conformation and stability of the monomer. Here we identify and characterize by protein and DNA analysis a novel amyloidogenic Val-20 to Ile mutation in a German three-generation family. The index patient suffered from severe amyloid cardiomyopathy at the age of 60. Conformational stability and unfolding behavior of the Ile-20 monomer in urea gradients was found to be almost indistinguishable from that of wild-type TTR. In contrast, tetramer stability was significantly reduced in agreement with the expected change in the interactions between the two opposing dimers via the side chain of Ile-20. Our observations provide strong evidence for the view that amyloidogenic amino acid substitutions in TTR facilitate the conversion of tetrameric TTR complexes into those conformational intermediates of the TTR folding pathway that have an intrinsic amyloidogenic potential.
Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.
Authors: Bensing B A , Meyer B J , Dunny G M
Published: July 2002

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (from prgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.
Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target.
Authors: Jakubczak J L , Merlino G , French J E , Muller W J , Paul B , Adhya S , Garges S
Published: July 2002

Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage lambda transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the lambda transgene that selects for phage containing forward mutations only in the lambda cII gene, using an hfl- bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, lambda lacI, making the cII assay an attractive alternative for mutant phage recovery for any lambda-based mouse mutagenesis assay system. The frequencies of lambda cII- mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases. The cII mutational spectra in these tissues consisted mostly of G/C-->A/T transitions, a large fraction of which occurred at CpG dinucleotides. These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.
A sequential dimerization mechanism for erythropoietin receptor activation.
Authors: Matthews D J , Topping R S , Cass R T , Giebel L B
Published: July 2002

We have probed the interaction of human erythropoietin (EPO) with its receptor (EPO-R) by analyzing a panel of 17 EPO mutants in a variety of in vitro assays. Mutant proteins were expressed in 293s cells and quantified by using an N-terminal epitope tag in conjunction with a surface plasmon resonance assay. Receptor binding was studied using both a soluble form of the EPO-R extracellular domain in an ELISA-format binding competition assay and full-length EPO-R in transfected BaF3 cells. Proliferative activity of the mutants was also determined in the BaF3-derived cell line and was correlated with the results from binding assays. Based on the results of these assays, we identified two distinct receptor binding sites on the EPO molecule. We propose that one site, containing residues Arg-150 and Lys-152, binds initially to EPO receptor on the cell surface. A second site, containing Arg-103 and Ser-104 (and possibly Arg-14), is involved in binding a second EPO-R at the cell surface, thus forming a homodimeric receptor complex. Furthermore, we demonstrate that one EPO mutant (R103A), which has previously been shown to lack proliferative function, is in fact an EPO antagonist. Taken together, these data support a sequential dimerization mechanism of EPO-R activation.
Forced expression of tropomyosin 2 or 3 in v-Ki-ras-transformed fibroblasts results in distinct phenotypic effects.
Authors: Gimona M , Kazzaz J A , Helfman D M
Published: July 2002

Transformation of cells in tissue culture results in a variety of cellular changes including alterations in cell growth, adhesiveness, motility, morphology, and organization of the cytoskeleton. Morphological and cytoskeletal changes are perhaps the most readily apparent features of transformed cells. Although a number of studies have documented a decrease in the expression of specific tropomyosin (TM) isoforms in transformed cells, it remains to be determined if the suppression of TM synthesis is essential in the establishment and maintenance of the transformed pheno-type. To address the roles of different TM isoforms in transformed cells we have examined the effects of expressing specific TM isoforms in transformed cells using a Kirsten virus-transformed cell line (ATCC NRK1569) as a model system. In contrast to normal fibroblasts, the NRK 1569 cells contain reduced levels of TM-1 and undetectable levels of TM-2 and TM-3. These cells have a rounded morphology and are devoid of stress fibers. Employing expression plasmids for TM-2 and TM-3, stable cell lines were established from the NRK 1569 cells that express these isoforms individually. We demonstrate that expression of TM-2 or TM-3 leads to increased cell spreading accompanied by the formation of identifiable microfilament bundles, as well as significant restoration of well-defined vinculin-containing focal adhesion plaques, although expression of each isoform exhibited distinct properties. In addition, cells expressing TM-2, but not TM-3, exhibited contact-inhibited cell growth and a requirement for serum.
A mechanism for the inhibition of fever by a virus.
Authors: Alcamí A , Smith G L
Published: July 2002

Poxviruses encode proteins that block the activity of cytokines. Here we show that the study of such virulence factors can contribute to our understanding of not only virus pathogenesis but also the physiological role of cytokines. Fever is a nonspecific response to infection that contributes to host defense. Several cytokines induce an elevation of body temperature when injected into animals, but in naturally occurring fever it has been difficult to show that any cytokine has a critical role. We describe the first example of the suppression of fever by a virus and the molecular mechanism leading to it. Several vaccinia virus strains including smallpox vaccines express soluble interleukin 1 (IL-1) receptors, which bind IL-1 beta but not IL-1 alpha. These viruses prevent the febrile response in infected mice, whereas strains that naturally or through genetic engineering lack the receptor induce fever. Repair of the defective IL-1 beta inhibitor in the smallpox vaccine Copenhagen, a more virulent virus than the widely used vaccine strains Wyeth and Lister, suppresses fever and attenuates the disease. The vaccinia-induced fever was inhibited with antibodies to IL-1 beta. These findings provide strong evidence that IL-1 beta, and not other cytokines, is the major endogenous pyrogen in a poxvirus infection.
Differences in water release for the binding of EcoRI to specific and nonspecific DNA sequences.
Authors: Sidorova N Y , Rau D C
Published: July 2002

The free energy difference between complexes of the restriction nuclease EcoRI with nonspecific DNA and with the enzyme's recognition sequence is linearly dependent on the water chemical potential of the solution, set using several very different solutes, ranging from glycine and glycerol to triethylene glycol and sucrose. This osmotic dependence indicates that the nonspecific complex sequesters some 110 waters more than the specific complex with the recognition sequence. The insensitivity of the difference in number of waters released to the solute identity further indicates that this water is sequestered in a space that is sterically inaccessible to solutes, most likely at the protein-DNA interface of the nonspecific complex. Calculations based on the structure of the specific complex suggest that the apposing DNA and protein surfaces in the nonspecific complex retain approximately a full hydration layer of water.
Alternative splicing of exon 3 of the human growth hormone receptor is the result of an unusual genetic polymorphism.
Authors: Stallings-Mann M L , Ludwiczak R L , Klinger K W , Rottman F
Published: July 2002

Two isoforms of the human growth hormone receptor (hGHR), which differ in the presence (hGHRwt) or absence (hGHRd3) of exon 3, are expressed in the placenta. Specifically, three expression patterns are observed: only hGHRwt, only hGHRd3, or an approximately 1:1 combination of both isoforms. We investigated several potential regulatory mechanisms which might account for the expression of the hGHR isoforms. The frequency of hGHRd3 expression did not change when placentas from differing stages of gestation were examined, suggesting splicing was not developmentally regulated. However, when hGHR isoform expression patterns were examined in each component of a given placenta, it was evident that alternative splicing of exon 3 is individual-specific. Surprisingly, the individual-specific regulation of hGHR isoforms appears to be the result of a polymorphism in the hGHR gene. We analyzed hGHRwt and hGHRd3 expression in Hutterite pedigrees, and our results are consistent with a simple Mendelian inheritance of two differing alleles in which exon 3 is spliced in an "all-or-none" fashion. We conclude the alternative splicing of exon 3 in hGHR transcripts is the result of an unusual polymorphism which significantly alters splicing of the hGHR transcript and that the relatively high frequency (approximately 10%) of homozygous hGHRd3 expression suggests the possibility it may play a role in polygenic determined events.
Large electrostatic differences in the binding thermodynamics of a cationic peptide to oligomeric and polymeric DNA.
Authors: Zhang W , Bond J P , Anderson C F , Lohman T M , Record M T
Published: July 2002

Results presented here demonstrate that the thermodynamics of oligocation binding to polymeric and oligomeric DNA are not equivalent because of long-range electrostatic effects. At physiological cation concentrations (0.1-0.3 M) the binding of an oligolysine octacation KWK6-NH2 (+8 charge) to single-stranded poly(dT) is much stronger per site and significantly more salt concentration dependent than the binding of the same ligand to an oligonucleotide, dT(pdT)10 (-10 charge). These large differences are consistent with Poisson-Boltzmann calculations for a model that characterizes the charge distributions with key preaveraged structural parameters. Therefore, both the experimental and the theoretical results presented here show that the polyelectrolyte character of a polymeric nucleic acid makes a large contribution to both the magnitude and the salt concentration dependence of its binding interactions with simple oligocationic ligands.
RecA protein stimulates homologous recombination in plants.
Authors: Reiss B , Klemm M , Kosak H , Schell J
Published: July 2002

A number of RecA-like proteins have been found in eukaryotic organisms. We demonstrate that the prokaryotic recombination protein RecA itself is capable of interacting with genomic homologous DNA in somatic plant cells. Resistance to the DNA crosslinking agent mitomycin C requires homologous recombination as well as excision repair activity. Tobacco protoplasts expressing a nucleus-targeted RecA protein were at least three times as efficient as wild-type cells in repairing mitomycin C-induced damage. Moreover, homologous recombination at a defined locus carrying an endogenous nuclear marker gene was stimulated at least 10-fold in transgenic plant cells expressing nucleus-targeted RecA. The increase in resistance to mitomycin C and the stimulation of intrachromosomal recombination demonstrate that Escherichia coli RecA protein is functional in genomic homologous recombination in plants, especially when targeted to the plant nucleus.
Human immunodeficiency virus (HIV) type 2-mediated inhibition of HIV type 1: a new approach to gene therapy of HIV-infection.
Authors: Arya S K , Gallo R C
Published: July 2002

Human immunodeficiency virus (HIV) type 2, the second AIDS-associated human retrovirus, differs from HIV-1 in its natural history, infectivity, and pathogenicity, as well as in details of its genomic structure and molecular behavior. We report here that HIV-2 inhibits the replication of HIV-1 at the molecular level. This inhibition was selective, dose-dependent, and nonreciprocal. The closely related simian immunodeficiency provirus also inhibited HIV-1. The selectivity of inhibition was shown by the observation that HIV-2 did not significantly downmodulate the expression of the unrelated murine leukemia virus; neither did the murine leukemia virus markedly affect HIV-1 or HIV-2 expression. Moreover, while HIV-2 potently inhibited HIV-1, the reverse did not happen, thus identifying yet another and remarkable difference between HIV-1 and HIV-2. Mutational analysis of the HIV-2 genome suggested that the inhibition follows a complex pathway, possibly involving multiple genes and redundant mechanisms. Introduction of inactivating mutations into the structural and regulatory/accessory genes did not render the HIV-2 provirus ineffective. Some of the HIV-2 gene defects, such as that of tat and rev genes, were phenotypically transcomplemented by HIV-1. The HIV-2 proviruses with deletions in the putative packaging signal and defective for virus replication were effective in inducing the suppressive phenotype. Though the exact mechanism remains to be defined, the inhibition appeared to be mainly due to an intracellular molecular event because it could not be explained solely on the basis of cell surface receptor mediated interference. The results support the notion that the inhibition likely occurred at the level of viral RNA, possibly involving competition between viral RNAs for some transcriptional factor essential for virus replication. Induction of a cytokine is another possibility. These findings might be relevant to the clinical-epidemiological data suggesting that infection with HIV-2 may offer some protection against HIV-1 infection.
Cloning of the gene for monogalactosyldiacylglycerol synthase and its evolutionary origin
Authors: Shimojima Mie , Ohta Hiroyuki , Iwamatsu Akihiro , Masuda Tatsuru , Shioi Yuzo , Takamiya Ken-ichiro
Published: July 2002

Monogalactosyldiacylglycerol (MGDG) synthase
(UDPgalactose:1,2-diacylglycerol
3-β-
d
-galactosyltransferase; EC
2.4.1.46
) catalyzes
formation of MGDG, a major structural lipid of chloroplast. We cloned a
cDNA for the synthase from cucumber cDNA library. The full-length cDNA
clone was 2142 bp, and it contains a 1575-bp open reading frame
encoding 525 aa. The open reading frame consists of the regions for a
mature protein (422 aa;
M
r
of 46,552) and
transit peptide to chloroplast (103 aa). Although the molecular weight
of mature protein region matched that purified from cucumber
cotyledons, it was quite different from those purified from spinach
(≈20 kDa) reported by other groups. The mature region of the protein
was expressed in
Escherichia coli
as a fusion protein
with glutathione
S
-transferase. The expression in
E. coli
showed that the protein catalyzed MGDG synthesis
very efficiently. Therefore, we concluded that the cDNA encodes MGDG
synthase in cucumber. In addition, the deduced amino acid sequence of
the MGDG synthase cDNA showed homology with MurG of
Bacillus
subtilis
and
E. coli
, which encode a
glycosyltransferase catalyzing the last step of peptidoglycan synthesis
in bacteria. This sequence homology implies that the machinery of
chloroplast membrane biosynthesis is evolutionarily derived from that
of cell wall biosynthesis in bacteria. This is consistent with the
endosymbiotic hypothesis of chloroplast formation.
Expression of maspin in prostate cells is regulated by a positive Ets element and a negative hormonal responsive element site recognized by androgen receptor
Authors: Zhang Ming , Magit David , Sager Ruth
Published: July 2002

Prostate cancer is the most common cancer in men. The molecular mechanisms leading to its development are poorly understood. Maspin is a tumor-suppressing serpin expressed in normal breast and prostate epithelium. We have found that expression of maspin in normal and carcinoma-derived prostate epithelial cells is differentially regulated at the transcriptional level. We have identified two different kinds of cis elements, Ets and hormonal responsive element (HRE), in the maspin promoter. The Ets element is active in regulating maspin expression in normal prostate epithelial cells but inactive in tumor cells. The HRE site is a negative element that is active in both cell types. This negative DNA sequence can repress a heterologous promoter recognized by the androgen receptor. We conclude that expression of maspin is under the influence of both a positive Ets and a negative HRE element. Loss of maspin expression during tumor progression apparently results from both the absence of transactivation through the Ets element and the presence of transcription repression through the negative HRE element recognized by androgen receptor.
Hydrogenase genes from
Rhizobium leguminosarum
bv. viciae are controlled by the nitrogen fixation regulatory protein NifA
Authors: Brito Belén , Martínez Marta , Fernández Domingo , Rey Luis , Cabrera Ezequiel , Palacios José Manuel , Imperial Juan , Ruiz-Argüeso Tomás
Published: July 2002

Rhizobium leguminosarum
bv. viciae expresses an uptake hydrogenase in symbiosis with peas (
Pisum sativum
) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene
hoxA
described in other species was shown to have been inactivated in
R. leguminosarum
by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes
hupSL
originates from a −24/−12 type promoter (
hupS
p
). A regulatory region located in the −173 to −88 region was essential for promoter activity in
R. leguminosarum
. Activation of
hupS
p
was observed in
Klebsiella pneumoniae
and
Escherichia coli
cells expressing the
K. pneumoniae
nitrogen fixation regulator NifA, and in
E. coli
cells expressing
R. meliloti
NifA. This activation required direct interaction of NifA with the essential −173 to −88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in
R. etli
bean bacteroids. NifA-dependent
hupS
p
activity in heterologous hosts was also absolutely dependent on the RpoN σ-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in
R. leguminosarum
. The data suggest that
hupS
p
is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H
2
production and H
2
oxidation in nodules might be the reason for the loss of HoxA in
R. leguminosarum
and the concomitant NifA control of
hup
gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H
2
-rich environment for rhizobia, the legume nodule.
Screening for imprinted genes by allelic message display: Identification of a paternally expressed gene
Impact
on mouse chromosome 18
Authors: Hagiwara Yuriko , Hirai Momoki , Nishiyama Kazutoshi , Kanazawa Ichiro , Ueda Takayuki , Sakaki Yoshiyuki , Ito Takashi
Published: July 2002

A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes. In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal F
1
hybrids and pooled backcross progenies. From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified. The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene
Impact
on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function. In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages. Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.
Independent mobility of catalytic and regulatory domains of myosin heads
Authors: Adhikari Bishow , Hideg Kalman , Fajer Piotr G.
Published: July 2002

The recent determination of the myosin head atomic structure has led to a new model of muscle contraction, according to which mechanical torque is generated in the catalytic domain and amplified by the lever arm made of the regulatory domain [Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995)
Biochemistry
34, 8960–8972]. A crucial aspect of this model is the ability of the regulatory domain to move independently of the catalytic domain. Saturation transfer–EPR measurements of mobility of these two domains in myosin filaments give strong support for this notion. The catalytic domain of the myosin head was labeled at Cys-707 with indane dione spin label; the regulatory domain was labeled at the single cysteine residue of the essential light chain and exchanged into myosin. The mobility of the regulatory domain in myosin filaments was characterized by an effective rotational correlation time (τ
R
) between 24 and 48 μs. In contrast, the mobility of the catalytic domain was found to be τ
R
= 5–9 μs. This difference in mobility between the two domains existed only in the filament form of myosin. In the monomeric form, or when bound to actin, the mobility of the two domains in myosin was indistinguishable, with τ
R
= 1–4 μs and >1,000 μs, respectively. Therefore, the observed difference in filaments cannot be ascribed to differences in local conformations of the spin-labeled sites. The most straightforward interpretation suggests a flexible hinge between the two domains, which would have to stiffen before force could be generated.
Calx, a Na-Ca exchanger gene ofDrosophila melanogaster
Authors: Schwarz Erich M. , Benzer Seymour
Published: July 2002

We have clonedCalx, a gene that encodes a Na-Ca exchanger ofDrosophila melanogaster.Calxencodes two repeated motifs,Calx-α andCalx-β, that overlap domains required for exchanger activity and regulation.Calxhas multiple transcripts in adults, including at least one expressed in the retina. TheCalxgenomic locus comprises ≥35 kb between theAtpαandrudimentary-likegenes in chromosomal region 93B. InXenopusoocytes, microinjectedCalxcRNA induces calcium uptake like that of its homolog, the 3Na+-1Ca2+exchanger of mammalian heart. Implications ofCalx-α motifs for the mechanism of Na-Ca exchange are discussed.
Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors
Authors: Murakami Katsuhiko , Owens Jeffrey T. , Belyaeva Tamara A. , Meares Claude F. , Busby Stephen J. W. , Ishihama Akira
Published: July 2002

Interactions between the cAMP receptor protein (CRP) and the carboxy-terminal regulatory domain (CTD) of
Escherichia coli
RNA polymerase α subunit were analyzed at promoters carrying tandem DNA sites for CRP binding using a chemical nuclease covalently attached to α. Each CRP dimer was found to direct the positioning of one of the two α subunit CTDs. Thus, the function of RNA polymerase may be subject to regulation through protein–protein interactions between the two α subunits and two different species of transcription factors.
Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2
Authors: Schlesinger Paul H. , Gross Atan , Yin Xiao-Ming , Yamamoto Kazuhito , Saito Mitsuyoshi , Waksman Gabriel , Korsmeyer Stanley J.
Published: July 2002

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-X
L
monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at ≈pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl
−
selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl
−
-selective (
P
K
/
P
Cl
= 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K
+
-selective (
P
K
/
P
Cl
= 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K
+
selectivity (
P
K
/
P
Cl
= 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.
Endoplasmic reticulum quality control of asialoglycoprotein receptor H2a involves a determinant for retention and not retrieval
Authors: Shenkman Marina , Ayalon Michal , Lederkremer Gerardo Z.
Published: July 2002

The human asialoglycoprotein receptor H2a subunit contains a charged pentapeptide, EGHRG, in its ectodomain that is the only sequence absent from the H2b alternatively spliced variant. H2b exits the endoplasmic reticulum (ER) even when singly expressed, whereas H2a gives rise to a cleaved soluble secreted ectodomain fragment; uncleaved membrane-bound H2a molecules are completely retained and degraded in the ER. We have inserted the H2a pentapeptide into the sequence of the H1 subunit (H1i5), which caused complete ER retention but, unexpectedly, no degradation. This suggests that the pentapeptide is a determinant for ER retention not colocalizing in H2a with the determinant for degradation. The state of sugar chain processing and the ER localization of H1i5, which was unchanged at 15°C or after treatment with nocodazole, indicate ER retention and not retrieval from the cis-Golgi or the intermediate compartment. H1i5 folded similarly to H1, and both associated to calnexin. However, whereas H1 dissociated with a half time of 45 min, H1i5 remained bound to the chaperone for prolonged periods. The correct global folding of H2a and H1i5 and of other normal precursors and unassembled proteins and the true ER retention, and not exit and retrieval, suggest a difference in their quality control mechanism compared with that of misfolded proteins, which does involve retrieval. However, both pathways may involve calnexin.