bluuebunny/crossref_metadata_2025 huggingface dataset:
Adjusting the binding thermodynamics, kinetics, and orientation of guests within large synthetic hydrophobic pockets
Authors: Gibb C. L. D. , Li X. , Gibb B. C.
Published: July 2002

Kinetic analysis of the host guest complexation of a large, open molecular basket and a highly complementary adamantoid guest reveals that for these types of systems a dissociative mechanism is in operation. Hence, the resident adamantyl guest must completely vacate the cavity before another guest molecule can move in to replace it. As a result of the rigid nature of the host, the energy barrier to this process is relatively high, about 16 kcal mol
−1
at room temperature. Modifying the cavity of the host by dangling either a methyl group or a hydroxyl group from the portal rim alters the thermodynamic binding profile of these hosts.
1
H NMR shift data analysis also reveals that these functional groups can adjust the orientation that monosubstituted guests adopt within the cavity. Additionally,
1
H NMR studies of the binding of (
E
)1,4-dibromoadamantane allow the observation of two energetically similar diastereomeric complexes. An examination of this guest binding to the three hosts reveals that the interchange between the isomers is much faster than the entry and egression rates, and that the functional groups at the rim of each cavity influence both the rates of reorientation and the equilibrium relating the isomers.
Endoplasmic reticulum localization of the low density lipoprotein receptor mediates presecretory degradation of apolipoprotein B
Authors: Gillian-Daniel Donald L. , Bates Paul W. , Tebon Angie , Attie Alan D.
Published: July 2002

Mutations in the low density lipoprotein (LDL) receptor (LDLR) cause hypercholesterolemia because of inefficient LDL clearance from the circulation. In addition, there is a paradoxical oversecretion of the metabolic precursor of LDL, very low density lipoprotein (VLDL). We recently demonstrated that the LDLR mediates pre-secretory degradation of the major VLDL protein, apolipoprotein B (apoB). Kinetic studies suggested that the degradation process is initiated in the secretory pathway. Here, we evaluated the ability of several LDLR variants that are stalled within the secretory pathway to regulate apoB secretion. Both a naturally occurring mutant LDLR and an LDLR consisting of only the ligand-binding domains and a C-terminal endoplasmic reticulum (ER) retention sequence were localized to the ER and not at the cell surface. In the presence of either of the ER-localized LDLRs, apoB secretion was essentially abolished. When the ligand-binding domain of the truncated receptor was mutated the receptor was unable to block apoB secretion, indicating that the inhibition of apoB secretion depends on the ability of the LDLR to bind to its ligand. These findings establish LDLR-mediated pre-secretory apoB degradation as a pathway distinct from reuptake of nascent lipoproteins at the cell surface. The LDLR provides an example of a receptor that modulates export of its ligand from the ER.
Minimal principle for rotor filaments
Authors: Wellner Marcel , Berenfeld Omer , Jalife José , Pertsov Arkady M.
Published: July 2002

Three-dimensional rotors, or scroll waves, provide essential insight into the activity of excitable media. They also are a suspected cause in the formation and maintenance of ventricular fibrillation, whose lethality is well known. It is therefore of considerable interest to find out what configurations can be adopted by such pathologies. A scroll's behavior is embodied in its organizing center or filament, a largely quiescent tube about which the scroll rotates. Predicting filament shape has normally required computer-intensive simulations of the whole scroll in time. We have found a fast and robust principle that yields the prediction for stationary filaments on a purely geometrical basis, blind to the reaction parameters of the medium. The procedure is to calculate the filament shape as a minimal path. We work in singly diffusive media whose diffusivity tensor—and no other feature—varies spatially. Mathematical and numerical evidence is presented for the proposition that a stable filament is a geodesic in a three-dimensional space whose metric is given by the inverse diffusivity tensor of the medium. Away from the boundaries, a stable filament is unaffected by the reaction parameters. The algorithmic aspects of this work are subsidiary to our main purpose of drawing attention to the universal and unexpectedly exact fit of an elementary geodesic principle within reaction–diffusion theories.
Hoxa 11is upstream ofIntegrinα8expression in the developing kidney
Authors: Valerius M. Todd , Patterson Larry T. , Feng Yuxin , Potter S. Steven
Published: July 2002

Mutation of the functionally redundantHoxa 11/Hoxd 11genes gives absent or rudimentary kidneys resulting from a dramatic reduction of the growth and branching of the ureteric bud. To understand better the molecular mechanisms ofHoxa 11/Hoxd 11function in kidney development, it is necessary to identify the downstream target genes regulated by their encoded transcription factors. To this end, we conducted a screen forHoxa 11-responsive genes in two kidney cell lines.HEK293cells, which usually do not expressHoxa 11, were modified to allow inducibleHoxa 11expression. ThemK10cells, derived specifically for this study fromHoxa 11/Hoxd 11double-mutant mice, were also modified to give cell populations with and withoutHoxa 11expression. Differential display, Gene Discovery Arrays, and Affymetrixgenechipprobe arrays were used to screen for genes up- or down-regulated byHoxa 11. Nine genes,PDGF A,Cathepsin L,annexin A1,Mm.112139,Est2 repressor factor,NrCAM,ZNF192,integrin-associated protein, andGCM1, showed reproducible 3-fold or smaller changes in gene expression in response toHoxa 11. One gene, theIntegrinα8, was up-regulated approximately 20-fold afterHoxa 11expression. TheIntegrinα8gene is expressed together withHoxa 11in metanephric mesenchyme cells, and mutation ofIntegrinα8gives a bud-branching morphogenesis defect very similar to that observed inHoxa 11/Hoxd 11mutant mice.In situhybridizations showed a dramatic regional reduction inIntegrinα8expression in the developing kidneys ofHoxa 11/Hoxd 11mutant mice. This work suggests that theIntegrinα8gene may be a major effector ofHoxa 11/Hoxd 11function in the developing kidney.
Genome complexity reduction for SNP genotyping analysis
Authors: Jordan Barbara , Charest Alain , Dowd John F. , Blumenstiel Justin P. , Yeh Ru-fang , Osman Asiah , Housman David E. , Landers John E.
Published: July 2002

Efficient single nucleotide polymorphism (SNP) genotyping methods are necessary to accomplish many current gene discovery goals. A crucial element in large-scale SNP genotyping is the number of individual biochemical reactions that must be performed. An efficient method that can be used to simultaneously amplify a set of genetic loci across a genome with high reliability can provide a valuable tool for large-scale SNP genotyping studies. In this paper we describe and characterize a method that addresses this goal. We have developed a strategy for reducing genome complexity by using degenerate oligonucleotide primer (DOP)-PCR and applied this strategy to SNP genotyping in three complex eukaryotic genomes; human, mouse, and
Arabidopsis thaliana
. Using a single DOP-PCR primer, SNP loci spread throughout a genome can be amplified and accurately genotyped directly from a DOP-PCR product mixture. DOP-PCRs are extremely reproducible. The DOP-PCR method is transferable to many species of interest. Finally, we describe an
in silico
approach that can effectively predict the SNP loci amplified in a given DOP-PCR, permitting the design of an efficient set of reactions for large-scale, genome-wide SNP studies.
The control of hematopoiesis and leukemia: from basic biology to the clinic.
Authors: Sachs L
Published: July 2002

Hematopoiesis gives rise to blood cells of different lineages throughout normal life. Abnormalities in this developmental program lead to blood cell diseases including leukemia. The establishment of a cell culture system for the clonal development of hematopoietic cells made it possible to discover proteins that regulate cell viability, multiplication and differentiation of different hematopoietic cell lineages, and the molecular basis of normal and abnormal blood cell development. These regulators include cytokines now called colony-stimulating factors (CSFs) and interleukins (ILs). There is a network of cytokine interactions, which has positive regulators such as CSFs and ILs and negative regulators such as transforming growth factor beta and tumor necrosis factor (TNF). This multigene cytokine network provides flexibility depending on which part of the network is activated and allows amplification of response to a particular stimulus. Malignancy can be suppressed in certain types of leukemic cells by inducing differentiation with cytokines that regulate normal hematopoiesis or with other compounds that use alternative differentiation pathways. This created the basis for the clinical use of differentiation therapy. The suppression of malignancy by inducing differentiation can bypass genetic abnormalities that give rise to malignancy. Different CSFs and ILs suppress programmed cell death (apoptosis) and induce cell multiplication and differentiation, and these processes of development are separately regulated. The same cytokines suppress apoptosis in normal and leukemic cells, including apoptosis induced by irradiation and cytotoxic cancer chemotherapeutic compounds. An excess of cytokines can increase leukemic cell resistance to cytotoxic therapy. The tumor suppressor gene wild-type p53 induces apoptosis that can also be suppressed by cytokines. The oncogene mutant p53 suppresses apoptosis. Hematopoietic cytokines such as granulocyte CSF are now used clinically to correct defects in hematopoiesis, including repair of chemotherapy-associated suppression of normal hematopoiesis in cancer patients, stimulation of normal granulocyte development in patients with infantile congenital agranulocytosis, and increase of hematopoietic precursors for blood cell transplantation. Treatments that decrease the level of apoptosis-suppressing cytokines and downregulate expression of mutant p53 and other apoptosis suppressing genes in cancer cells could improve cytotoxic cancer therapy. The basic studies on hematopoiesis and leukemia have thus provided new approaches to therapy.
In vivo suppression of the renal Na+/Pi cotransporter by antisense oligonucleotides.
Authors: Oberbauer R , Schreiner G F , Biber J , Murer H , Meyer T W
Published: July 2002

A 20-mer phosphorothioate oligonucleotide (AS1) was designed to hybridize to the message for the rat kidney sodium phosphate cotransporter NaPi-2 close to the translation initiation site. Single intravenous doses of this oligonucleotide were given to rats maintained on a low phosphorus diet to increase NaPi-2 expression. At 3 days after oligonucleotide infusion, rats receiving 2.5 micromol of AS1 exhibited a reduction in renal NaPi-2 to cyclophilin mRNA ratio by 40% +/- 17%, and rats receiving 7.5 micromol of AS1 exhibited a reduction in NaPi-2 to cyclophilin mRNA ratio by 46% +/- 21%. Reversed-sequence AS1 was without effect. The higher dose of 7.5 micromol of AS1 also reduced the rate of phosphate uptake into renal brush border membrane vesicles and the expression of NaPi-2 protein detected by Western blotting in these vesicles. Reversed sequence AS1 was again without effect on these parameters. These results suggest that systemically infused oligonucleotides can exert antisense effects in the renal proximal tubule.
Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.
Authors: Dobie K W , Lee M , Fantes J A , Graham E , Clark A J , Springbett A , Lathe R , McClenaghan M
Published: July 2002

Mice carrying an ovine beta-lactoglobulin (BLG) transgene secrete BLG protein into their milk. To explore transgene expression stability, we studied expression levels in three BLG transgenic mouse lines. Unexpectedly, two lines exhibited variable levels of transgene expression. Copy number within lines appeared to be stable and there was no evidence of transgene rearrangement. In the most variable line, BLG production levels were stable within individual mice in two successive lactations. Backcrossing demonstrated that genetic background did not contribute significantly to variable expression. Tissue in situ hybridization revealed mosaicism of transgene expression within individual mammary glands from the two variable lines; in low expressors, discrete patches of cells expressing the transgene were observed. Transgene protein concentrations in milk reflected the proportion of epithelial cells expressing BLG mRNA. Furthermore, chromosomal in situ hybridization revealed that transgene arrays in both lines are situated close to the centromere. We propose that mosaicism of transgene expression is a consequence of the chromosomal location and/or the nature of the primary transgene integration event.
p34cdc2 kinase activity is maintained upon activation of the replication checkpoint in Schizosaccharomyces pombe.
Authors: Knudsen K E , Knudsen E S , Wang J Y , Subramani S
Published: July 2002

All eukaryotes use feedback controls to order and coordinate cell cycle events. In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis. Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear. Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase. It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically. We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor. Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint. We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast. Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity.
Proliferation of multipotent hematopoietic cells controlled by a truncated erythropoietin receptor transgene.
Authors: Kirby S L , Cook D N , Walton W , Smithies O
Published: July 2002

The long-term efficacy of gene therapy using bone marrow transplantation requires the engraftment of genetically altered totipotent hematopoietic stem cells (THSCs). Ex vivo expansion of corrected THSCs is one way to increase the efficiency of the procedure. Similarly, selective in vivo expansion of the therapeutic THSCs rather than the endogenous THSCs could favor the transplant. To test whether a conferred proliferative advantage gene can facilitate the in vitro and in vivo expansion of hematopoietic stem cells, we have generated transgenic mice expressing a truncated receptor for the growth factor erythropoietin. These mice are phenotypically normal, but when treated in vivo with exogenous erythropoietin they exhibit a marked increase in multipotent, clonogenic hematopoietic cells [colony-forming units in the spleen (CFU-S) and CFUs that give rise to granulocytes, erythroid cells, macrophages, and megakaryocytes within the same colony (CFU-GEMM)] in comparison with the wild-type mice. In addition, long-term in vitro culture of tEpoR transgenic bone marrow in the presence of erythropoietin induces exponential expansion of trilineage hematopoietic stem cells not seen with wild-type bone marrow. Thus, the truncated erythropoietin receptor gene shows promise as a means for obtaining cytokine-inducible hematopoietic stem cell proliferation to facilitate the direct targeting of THSCs and to provide a competitive repopulation advantage for transplanted therapeutic stem cells.
Organization and chromosomal localization of the gene encoding the mouse acid labile subunit of the insulin-like growth factor binding complex.
Authors: Boisclair Y R , Seto D , Hsieh S , Hurst K R , Ooi G T
Published: July 2002

After birth, most of insulin-like growth factor I and II (IGFs) circulate as a ternary complex formed by the association of IGF binding protein 3-IGF complexes with a serum protein called acid-labile subunit (ALS). ALS retains the IGF binding protein-3-IGF complexes in the vascular compartment and extends the t1/2 of IGFs in the circulation. Synthesis of ALS occurs mainly in liver after birth and is stimulated by growth hormone. To study the basis for this regulation, we cloned and characterized the mouse ALS gene. Comparison of genomic and cDNA sequences indicated that the gene is composed of two exons separated by a 1126-bp intron. Exon 1 encodes the first 5 amino acids of the signal peptide and contributes the first nucleotide of codon 6. Exon 2 contributes the last 2 nt of codon 6 and encodes the remaining 17 amino acids of the signal peptide as well as the 580 amino acids of the mature protein. The polyadenylylation signal, ATTAAA, is located 241 bp from the termination codon. The cDNA and genomic DNA diverge 16 bp downstream from this signal. Transcription initiation was mapped to 11 sites over a 140-bp TATA-less region. The DNA fragment extending from nt -805 to -11 (ATG, +1) directed basal and growth hormone-regulated expression of a luciferase reporter plasmid in the rat liver cell line H4-II-E. Finally, the ALS gene was mapped to mouse chromosome 17 by fluorescence in situ hybridization.
In situ
molecular association of dystrophin with actin revealed by sensitized emission immuno-resonance energy transfer
Authors: Root Douglas D.
Published: July 2002

A novel method was developed to detect molecular associations of dystrophin with actin in cryostat muscle tissue sections by combining resonance energy transfer technology with immunohistochemical techniques. This method takes advantage of the long phosphorescent lifetime of terbium chelates, a property that enables the accurate determination of energy transfer in biological tissues by lifetime measurements of sensitized emission. After a brief excitation pulse, terbium chelates emit for milliseconds after the intrinsically high autofluorescence of biological specimens has decayed to negligible levels. Rat skeletal muscle tissue sections were labeled with both anti-dystrophin monoclonal antibody conjugated to a terbium-based resonance energy transfer donor and anti-actin tetramethylrhodamine phalloidin as an acceptor. Resonance energy transfer between the two probes indicated that the distance separating the probes is within 10 nm (about the size of an IgG
2b
antibody molecule). The fraction of antibodies that participated in resonance energy transfer was estimated to be 80–90% because of the close agreement between the quenching of donor phosphorescence and the efficiency of resonance energy transfer revealed by lifetime measurements of sensitized emission by tetramethyl-rhodamine phalloidin. Sensitized emission was detectable only when both anti-dystrophin antibody and tetramethyl-rhodamine phalloidin were present. These results indicate that actin and dystrophin are closely associated within the cell. This method is potentially applicable to the investigation of many types of intracellular associations.
ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques
Authors: Robinson Lois , Panayiotakis Alexandra , Papas Takis S. , Kola Ismail , Seth Arun
Published: July 2002

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A
2
-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with
Mbo
I-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.
Demonstration of a novel circulating anti-prostacyclin receptor antibody
Authors: Kahn Nighat N. , Bauman William A. , Sinha Asru K.
Published: July 2002

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI
2
) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (
n
= 12) also resulted in the loss of high-affinity binding of PGI
2
(
Kd
1
= 9.1 ± 2.0 nM;
n
1
= 170 ± 32 sites per cell vs.
Kd
1
= 7.2 ± 1.1 nM;
n
1
= 23 ± 8 sites per cell), with no significant change in the low-affinity receptors (
Kd
2
= 1.9 ± 0.1 μM;
n
2
= 1,832 ± 232 sites per cell vs.
Kd
2
= 1.6 ± 0.1 μM;
n
2
= 1,740 ± 161 sites per cell) as determined by Scatchard analysis of the binding of [
3
H]PGE
1
. The loss of high-affinity PGI
2
binding led to the failure of PGI
2
to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI
2
to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [
3
H]PGE
1
to the high-affinity PGI
2
receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [
3
H]PGE
1
to the high-affinity platelet PGI
2
receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI
2
binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.
Converting a transmembrane receptor to a soluble receptor: Recognition domain to effector domain signaling after excision of the transmembrane domain
Authors: Ottemann Karen M. , Koshland Daniel E.
Published: July 2002

The bacterial aspartate receptor was reconstructed to eliminate the transmembrane domain, thus connecting the recognition domain directly to the effector domain. The resulting soluble receptor folded correctly and was no longer an integral membrane protein. Upon aspartate binding, this soluble receptor was stabilized to a similar extent as that of the native receptor. Of interest, this soluble receptor retained the ability to signal from the recognition to the effector domain. This result defines more clearly the role of the membrane and transmembrane domains in signal transduction and suggests that some ligand-induced motions in receptor proteins do not require the membrane or transmembrane domain for information transmission.
Silencer elements controlling the B29 (Igβ) promoter are neither promoter- nor cell-type-specific
Authors: Malone Cindy Sue , Omori Sidne A. , Wall Randolph
Published: July 2002

The murine B29 (Igβ) promoter is B cell specific and contains essential SP1, ETS, OCT, and Ikaros motifs. Flanking 5′ DNA sequences inhibit B29 promoter activity, suggesting this region contains silencer elements. Two adjacent 5′ DNA segments repress transcription by the murine B29 promoter in a position- and orientation-independent manner, analogous to known silencers. Both these 5′ segments also inhibit transcription by several heterologous promoters in B cells, including mb-1, c-fos,and human B29. These 5′ segments also inhibit transcription by the c-fospromoter in T cells suggesting they are not B cell-specific elements. DNase I footprint analyses show an approximately 70-bp protected region overlapping the boundary between the two negative regulatory DNA segments and corresponding to binding sites for at least two different DNA-binding proteins. Within this footprint, two unrelated 30-bp cis-acting DNA motifs (designated TOAD and FROG) function as position- and orientation-independent silencers when located directly 5′ of the murine B29 promoter. These two silencer motifs act cooperatively to restrict the transcriptional activity of the B29 promoter. Neither of these motifs resembles any known silencers. Mutagenesis of the TOAD and FROG motifs in their respective 5′ DNA segments eliminates the silencing activity of these upstream regions, indicating these two motifs as the principal B29 silencer elements within these regions.
The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon
Pyrococcus furiosus
, a missing link in the evolution of carbamoyl phosphate biosynthesis
Authors: Durbecq Virginie , Legrain Christianne , Roovers Martine , Piérard André , Glansdorff Nicolas
Published: July 2002

Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon
Pyrococcus furiosus
was found to synthesize CP by using ammonia and not glutamine. We have purified the cognate enzyme and found it to be a dimer of two identical subunits of
M
r
32,000. Its thermostability is considerable, 50% activity being retained after 1 h at 100°C or 3 h at 95°C. The corresponding gene was cloned by PCR and found to present about 50% amino acid identity with known CKs. The stoichiometry of the reaction (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish
P. furiosus
CPS from ordinary CKs. Thus the CPS of
P. furiosus
could represent a primeval step in the evolution of CPS from CK. Our results suggest that the first event in this evolution was the emergence of a primeval synthetase composed of subunits able to synthesize both carboxyphosphate and CP; this step would have preceded the duplication assumed to have generated the two subdomains of modern CPSs. The gene coding for this CK-like CPS was called
cpkA
.
Selective activation of JNK1 is necessary for the anti-apoptotic activity of hILP
Authors: Sanna M. Germana , Duckett Colin S. , Richter Bettina W. M. , Thompson Craig B. , Ulevitch Richard J.
Published: July 2002

The balance between the inductive signals and endogenous anti-apoptotic mechanisms determines whether or not programmed cell death occurs. The widely expressed inhibitor of apoptosis gene family includes three closely related mammalian proteins: c-IAP1, c-IAP2, and hILP. The anti-apoptotic properties of these proteins have been linked to caspase inhibition. Here we show that one member of this group, hILP, inhibits interleukin-1β-converting enzyme-induced apoptosis via a mechanism dependent on the selective activation of c-Jun N-terminal kinase 1. These data demonstrate that apoptosis can be inhibited by an endogenous cellular protein by a mechanism that requires the activation of a single member of the mitogen-activating protein kinase family.
Model studies directed toward the application of boron neutron capture therapy to rheumatoid arthritis: Boron delivery by liposomes in rat collagen-induced arthritis
Authors: Watson-Clark Rachel A. , Banquerigo Mona Lisa , Shelly Kenneth , Hawthorne M. Frederick , Brahn Ernest
Published: July 2002

The application of boron neutron capture therapy to rheumatoid arthritis requires the selective delivery of the boron-10 isotope to the synovitic tissue. The use of liposomes as a boron delivery method has been explored through the measurement of the time course biodistribution of boron in rats with collagen-induced arthritis (CIA). Small unilamellar vesicles were composed of a 1:1 mixture of distearoylphosphatidylcholine and cholesterol, incorporated K[
nido
-7-CH
3
(CH
2
)
15
-7,8-C
2
B
9
H
11
] as an addend in the lipid bilayer and encapsulated Na
3
[
a
2
-B
20
H
17
NH
2
CH
2
CH
2
NH
2
] in the aqueous core. The tissue concentration of boron delivered by liposomes was determined by inductively coupled plasma–atomic emission spectroscopy after intravenous injection of liposome suspensions into Louvain rats with CIA. With the low injected doses of boron used [13–18 mg of boron per kg (body weight)], the peak boron concentration observed in arthritic synovium was 29 μg of boron per g of tissue. The highest synovium/blood boron ratio observed was 3.0, when the synovial boron concentration was 22 μg of boron per g of tissue. In an attempt to increase the synovium/blood boron ratio by lowering the blood boron concentration, a liposomal formulation characterized by a shorter blood clearance time was examined. Thus, the biodistribution of liposomes with additional K[
nido
-7-CH
3
(CH
2
)
15
-7,8-C
2
B
9
H
11
] incorporated in the vesicle membrane not only demonstrated more rapid blood clearance and slightly higher synovium/blood boron ratios but also exhibited reduced boron uptake in synovial tissue. These studies with boron neutron capture therapy for CIA suggest that this form of therapy may be feasible in the treatment of rheumatoid arthritis.
Tritium planigraphy: From the accessible surface to the spatial structure of a protein
Authors: Bogacheva Elena N. , Gol’danskii Vitalii I. , Shishkov Alexander V. , Galkin Alexander V. , Baratova Ludmila A.
Published: July 2002

The method of tritium planigraphy, which provides comprehensive information on the accessible surface of macromolecules, allows an attempt at reconstructing the three-dimensional structure of a protein from the experimental data on residue accessibility for labeling. The semiempirical algorithm proposed for globular proteins involves (
i
) predicting theoretically the secondary structure elements (SSEs), (
ii
) experimentally determining the residue-accessibility profile by bombarding the whole protein with a beam of hot tritium atoms, (
iii
) generating the residue-accessibility profiles for isolated SSEs by computer simulation, (
iv
) locating the contacts between SSEs by collating the experimental and simulated accessibility profiles, and (
v
) assembling the SSEs into a compact model via these contact regions in accordance with certain rules. For sperm whale myoglobin, carp and pike parvalbumins, the λ
cro
repressor, and hen egg lysozyme, this algorithm yields the most realistic models when SSEs are assembled sequentially from the amino to the carboxyl end of the protein chain.
Stimulation of p70S6 kinase via a growth hormone-controlled phosphatidylinositol 3-kinase pathway leads to the activation of a PDE4A cyclic AMP-specific phosphodiesterase in 3T3-F442A preadipocytes
Authors: MacKenzie Simon J. , Yarwood Stephen J. , Peden Alexander H. , Bolger Graeme B. , Vernon Richard G. , Houslay Miles D.
Published: July 2002

The challenge of 3T3-F442A fibroblasts with growth hormone led to both a decrease in the mobility on SDS/PAGE and activation of the PDE4A cyclic AMP-specific phosphodiesterase isoform PDE4A5. Activation was mediated by a JAK-2-dependent pathway coupled to the activation of phosphatidylinositol 3-kinase and p70S6 kinase. Activation was not dependent on the ability of growth hormone to stimulate ERK2 or protein kinase C or any effect on transcription. Blockade of activation of murine PDE4A5 ablated the ability of growth hormone to decrease intracellular cAMP levels. Antisense depletion of murine PDE4A5 mimicked the ability of rolipram to enhance the growth hormone-stimulated differentiation of 3T3-F442A cells to adipocytes. It is suggested that activation of PDE4A5 by growth hormone serves as a brake on the differentiation processes.
Rapid isolation of cDNA by hybridization
Authors: Hamaguchi Masaaki , O’Connor Elizabeth A. , Chen Tong , Parnell Larry , McCombie Richard W. , Wigler Michael H.
Published: July 2002

The isolation of genes from a given genomic region can be a rate-limiting step in the discovery of disease genes. We describe an approach to the isolation of cDNAs that have sequences in common with large genomic clones such as bacterial artificial chromosomes. We applied this method to loci both amplified and deleted in cancer, illustrating its usage in the identification of both oncogenes and tumor suppressor genes, respectively. The method, called rapid isolation of cDNAs by hybridization (RICH), depends on solution hybridization, enzymatic modification, and amplification/selection of sequences present in both cDNA populations and the genomic clones. The method should facilitate the development of transcription maps for large genomic clones, possibly even yeast artificial chromosomes.
Different mechanisms for suppression of apoptosis by cytokines and calcium mobilizing compounds
Authors: Lotem Joseph , Sachs Leo
Published: July 2002

Overexpression of wild-type p53 in M1 myeloid leukemia cells induces apoptotic cell death that was suppressed by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin (TG). This suppression of apoptosis by A23187 or TG was associated with suppression of caspase activation but not with suppression of wild-type-p53-induced expression ofWAF-1, mdm-2,orFAS. In contrast to suppression of apoptosis by the cytokines interleukin 6 (IL-6) and interferon γ, a protease inhibitor, or an antioxidant, suppression of apoptosis by A23187 or TG required extracellular Ca2+and was specifically abolished by the calcineurin inhibitor cyclosporin A. IL-6 induced immediate early activation ofjunBandzif/268 (Egr-1) but A23187 and TG did not. A23187 and TG also suppressed induction of apoptosis by doxorubicin or vincristine in M1 cells that did not express p53 by a cyclosporin A-sensitive mechanism. Suppression of apoptosis by A23187 or TG was not associated with autocrine production of IL-6. Apoptosis induced in IL-6-primed M1 cells after IL-6 withdrawal was not suppressed by A23187 or TG but was suppressed by the cytokines IL-6, IL-3, or interferon γ. The results indicate that these Ca2+-mobilizing compounds can suppress some pathways of apoptosis suppressed by cytokines but do so by a different mechanism.
The cytosolic tail of class I MHC heavy chain is required for its dislocation by the human cytomegalovirus US2 and US11 gene products
Authors: Story Craig M. , Furman Margo H. , Ploegh Hidde L.
Published: July 2002

The US2 and US11 glycoproteins of human cytomegalovirus facilitate destruction of MHC class I heavy chains by proteasomal proteolysis through acceleration of endoplasmic reticulum-to-cytosol dislocation. Modification of the class I heavy chain was used to probe the structural requirements for this sequence of reactions. The cytosolic domain of the class I heavy chain is required for dislocation to the cytosol and for its subsequent destruction. However, interactions between US2 or US11 and the heavy chain are maintained in the absence of the class I cytosolic domain, as shown by chemical crosslinkingin vivoand coprecipitation when translatedin vitro. Thus, substrate recognition and accelerated destruction of the heavy chain, as facilitated by US2 or US11, are separable events.
Proton uptake by bacterial reaction centers: The protein complex responds in a similar manner to the reduction of either quinone acceptor
Authors: Miksovska J. , Schiffer M. , Hanson D. K. , Sebban P.
Published: July 2002

In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone Q
A
−
and the secondary acceptor Q
B
−
after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near Q
B
; some of them carry additional mutations distant from the quinone sites (M231Arg → Leu, M43Asn → Asp, M5Asn → Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala → Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either Q
A
−
or Q
B
−
, suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to Q
B
is evident from examination of the pattern of H
+
/Q
A
−
proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q
−
species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.
Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain
Authors: Nikolskaya T. , Zagnitko O. , Tevzadze G. , Haselkorn R. , Gornicki P.
Published: July 2002

A series of chimeral genes, consisting of the yeast
GAL10
promoter, yeast
ACC1
leader, wheat acetyl-CoA carboxylase (ACCase; EC
6.4.1.2
) cDNA, and yeast
ACC1
3′-tail, was used to complement a yeast
ACC1
mutation. These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins. Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5′-end, complement the yeast mutation. Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 μM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases. This inhibition resembles the inhibition of wheat plastid ACCase observed
in vitro
and
in vivo
. The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase. Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors. The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors.
Nucleic acid duplexes incorporating a dissociable covalent base pair
Authors: Gao Kui , Orgel Leslie E.
Published: July 2002

We have used molecular modeling techniques to design a dissociable covalently bonded base pair that can replace a Watson-Crick base pair in a nucleic acid with minimal distortion of the structure of the double helix. We introduced this base pair into a potential precursor of a nucleic acid double helix by chemical synthesis and have demonstrated efficient nonenzymatic template-directed ligation of the free hydroxyl groups of the base pair with appropriate short oligonucleotides. The nonenzymatic ligation reactions, which are characteristic of base paired nucleic acid structures, are abolished when the covalent base pair is reduced and becomes noncoplanar. This suggests that the covalent base pair linking the two strands in the duplex is compatible with a minimally distorted nucleic acid double-helical structure.
MEKK1 suppresses oxidative stress-induced apoptosis of embryonic stem cell-derived cardiac myocytes
Authors: Minamino Tetsuo , Yujiri Toshiaki , Papst Philip J. , Chan Edward D. , Johnson Gary L. , Terada Naohiro
Published: July 2002

A combination of
in vitro
embryonic stem (ES) cell differentiation and targeted gene disruption has defined complex regulatory events underlying oxidative stress-induced cardiac apoptosis, a model of postischemic reperfusion injury of myocardium. ES cell-derived cardiac myocytes (ESCM) having targeted disruption of the MEKK1 gene were extremely sensitive, relative to wild-type ESCM, to hydrogen peroxide-induced apoptosis. In response to oxidative stress, MEKK1−/− ESCM failed to activate c-Jun kinase (JNK) but did activate p38 kinase similar to that observed in wild-type ESCM. The increased apoptosis was mediated through enhanced tumor necrosis factor α production, a response that was positively and negatively regulated by p38 and the MEKK1-JNK pathway, respectively. Thus, MEKK1 functions in the survival of cardiac myocytes by inhibiting the production of a proapoptotic cytokine. MEKK1 regulation of the JNK pathway is a critical response for the protection against oxidative stress-induced apoptosis in cardiac myocytes.
Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells
Authors: Chen Fang , Figueroa David J. , Marmorstein Alan D. , Zhang Qing , Petrukhin Konstantin , Caskey C. Thomas , Austin Christopher P.
Published: July 2002

In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999)
Proc. Natl. Acad. Sci. USA
96, 4814–4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription–PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of ≈7.5 kb, ≈3.0 kb, and ≈2.3 kb by Northern blot analysis.
In situ
hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.
Yeast and human genes that affect the
Escherichia coli
SOS response
Authors: Perkins Edward L. , Sterling Joan F. , Hashem Vera I. , Resnick Michael A.
Published: July 2002

The sequencing of the human genome has led to the identification of many genes whose functions remain to be determined. Because of conservation of genetic function, microbial systems have often been used for identification and characterization of human genes. We have investigated the use of the
Escherichia coli
SOS induction assay as a screen for yeast and human genes that might play a role in DNA metabolism and/or in genome stability. The SOS system has previously been used to analyze bacterial and viral genes that directly modify DNA. An initial screen of meiotically expressed yeast genes revealed several genes associated with chromosome metabolism (e.g.,
RAD51
and
HHT1
as well as others). The SOS induction assay was then extended to the isolation of human genes. Several known human genes involved in DNA metabolism, such as the Ku70 end-binding protein and DNA ligase IV, were identified, as well as a large number of previously unknown genes. Thus, the SOS assay can be used to identify and characterize human genes, many of which may participate in chromosome metabolism.
Overexpression of a Shaker-type potassium channel in mammalian central nervous system dysregulates native potassium channel gene expression
Authors: Sutherland M. L. , Williams S. H. , Abedi R. , Overbeek P. A. , Pfaffinger P. J. , Noebels J. L.
Published: July 2002

The nervous system maintains a delicate balance between excitation and inhibition, partly through the complex interplay between voltage-gated sodium and potassium ion channels. Because K+channel blockade or gene deletion causes hyperexcitability, it is generally assumed that increases in K+channel gene expression should reduce neuronal network excitability. We have tested this hypothesis by creating a transgenic mouse that expresses a Shaker-type K+channel gene. Paradoxically, we find that addition of the extra K+channel gene results in a hyperexcitable rather than a hypoexcitable phenotype. The presence of the transgene leads to a complex deregulation of endogenous Shaker genes in the adult central nervous system as well as an increase in network excitability that includes spontaneous cortical spike and wave discharges and a lower threshold for epileptiform bursting in isolated hippocampal slices. These data suggest that an increase in K+channel gene dosage leads to dysregulation of normal K+channel gene expression, and it may underlie a mechanism contributing to the pathogenesis of human aneuploidies such as Down syndrome.