bluuebunny/crossref_metadata_2025 huggingface dataset:
Newly expressed progesterone receptor cannot activate stable, replicated mouse mammary tumor virus templates but acquires transactivation potential upon continuous expression.
Authors: Smith C L , Archer T K , Hamlin-Green G , Hager G L
Published: May 2006

During development and differentiation, the expression of transcription factors is regulated in a temporal fashion. Newly expressed transcription factors must interact productively with target genes organized in chromatin. Although the mechanisms governing factor binding to chromatin templates are not well understood, it is now clear that template access can be dramatically influenced by nucleoprotein structure. We have examined the ability of a well characterized transactivator, the progesterone receptor (PR), to activate the mouse mammary tumor virus (MMTV) promoter organized either in stable, replicating templates that have a highly ordered nucleosome structure or as transiently transfected DNA, which adopts a less-defined structure. If the PR is transiently expressed in cells harboring both replicated and transient MMTV receptor constructs, it cannot significantly activate the stable replicated MMTV template. In contrast, when PR cDNA is stably inserted into the same cells and constitutively expressed, it gains the ability to activate both chromosomal and transiently introduced templates. These results demonstrate that newly expressed PR is not competent to activate the MMTV template in its native nucleoprotein conformation but acquires this ability upon prolonged expression in replicating cells.
Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II.
Authors: Kretzschmar M , Meisterernst M , Roeder R G
Published: May 2006

The transcriptional activation of eukaryotic class II genes by sequence-specific regulatory proteins requires cofactors in addition to the general transcription factors. One cofactor (termed PC3) was purified from HeLa cells and identified by sequence analysis and functional assays as human DNA topoisomerase I (EC5.99.1.2). Under identical conditions PC3 mediates both a net activation of transcription by the acidic activator GAL4-AH and repression of basal transcription, thereby leading to a large induction of transcription by the activator. PC3-mediated activation of transcription is dependent on the presence of both the GAL4-AH activation domain and the TATA-binding protein (TBP)-associated-factors (TAFs) in natural transcription factor TFIID, while repression of basal transcription is observed with either TFIID or the derived TBP alone. These results suggest novel functions, apparently through distinct mechanisms, for human DNA topoisomerase I in the regulation of transcription initiation by RNA polymerase II.
V3 variability can influence the ability of an antibody to neutralize or enhance infection by diverse strains of human immunodeficiency virus type 1.
Authors: Kliks S C , Shioda T , Haigwood N L , Levy J A
Published: May 2006

Human monoclonal antibodies (mAbs) to two contiguous epitopes in the V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope have shown different effects on three distinct strains of the virus: neutralization, enhancement, or resistance to both processes. Only one amino acid in the mAb epitopes proximal to the crown of the V3 loop was different among these three strains. Substitution of this amino acid in the neutralizable strain with the amino acid of the neutralization-resistant strain or the enhanceable strain resulted in loss of both activities. The conversion of this single amino acid in the neutralization-resistant strain to that of the amino acid found in the neutralization-sensitive strain did not confer the ability for the virus to be neutralized. However, additional changes in neighboring amino acids in the V3 loop succeeded in conferring the neutralization capability. These observations indicate that one antibody species can exert three different effects on various HIV-1 strains. They could explain the emergence of neutralization "escape" variants in the presence of the neutralizing antibodies. Moreover, the results suggest caution in immunization of individuals with the envelope region from one strain since the antibodies induced may show a neutralizing effect against the homologous strain but enhancing effects against other unrelated strains.
Osteoporosis induced in mice by overproduction of interleukin 4.
Authors: Lewis D B , Liggitt H D , Effmann E L , Motley S T , Teitelbaum S L , Jepsen K J , Goldstein S A , Bonadio J , Carpenter J , Perlmutter R M
Published: May 2006

Osteoporosis is a common disease in which loss of bone mass results in skeletal fragility. The development of therapies for this disorder has been hampered by the lack of a convenient animal model. Here we describe a disorder in bone homeostasis in transgenic mice that inappropriately express the cytokine interleukin 4 (IL-4) under the direction of the lymphocyte-specific proximal promoter for the lck gene. Bone disease in lck-IL-4 mice appeared to result from markedly decreased bone formation by osteoblasts, features strikingly similar to those observed in cases of severe low-turnover human involutional osteoporosis. By 2 months of age, female and male lck-IL-4 mice invariably developed severe osteoporosis of both cortical and trabecular bone. Osteoporosis was observed in two independently derived founder animals, indicating that this phenotype was directly mediated by the IL-4 transgene.
The mdm-2 gene is induced in response to UV light in a p53-dependent manner.
Authors: Perry M E , Piette J , Zawadzki J A , Harvey D , Levine A J
Published: May 2006

Irradiation of mammalian cells with UV light results in a dose-dependent accumulation of the p53 tumor-suppressor gene product that is evident within 2 hr. UV treatment causes a dramatic increase in p53-specific transcriptional transactivation activity and an increase in expression of the p53-responsive gene mdm-2. UV-stimulated mdm-2 expression is not directly correlated with the level of p53 protein in a cell because mdm-2 induction is delayed at high UV doses even though p53 levels rise almost immediately. Cells lacking p53 protein do not respond to UV by increasing their expression of mdm-2. The delayed induction of mdm-2 at high UV doses suggests that, in addition to p53 protein levels, other factors contribute to the regulation of mdm-2 expression following UV treatment. The time of induction of mdm-2 in cells treated with UV light correlates with recovery of normal rates of DNA synthesis, presumably after DNA repair. These data indicate a possible role for mdm-2 in cell cycle progression.
Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6.
Authors: Tamura T , Udagawa N , Takahashi N , Miyaura C , Tanaka S , Yamada Y , Koishihara Y , Ohsugi Y , Kumaki K , Taga T
Published: May 2006

It has been reported that soluble interleukin (IL)-6 receptor (sIL-6R) is detected in the serum of healthy individuals and its level is increased in patients with multiple myeloma and human immunodeficiency virus infection. Although several reports have suggested that sIL-6R potentiates IL-6 action, its physiological role remains unclear. In this study, we examined the role of sIL-6R on osteoclast formation by IL-6, using a coculture of mouse osteoblasts and bone marrow cells. Neither recombinant mouse IL-6 (mIL-6) nor mouse sIL-6R (smIL-6R) induced osteoclast-like multinucleated cell (MNC) formation when they were added separately. In contrast, simultaneous treatment with mIL-6 and smIL-6R strikingly induced MNC formation. These MNCs satisfied major criteria of authentic osteoclasts, such as tartrate-resistant acid phosphatase (TRAP) activity, calcitonin receptors, and pit formation on dentine slices. The MNC formation induced by mIL-6 and smIL-6R was dose-dependently inhibited by adding monoclonal anti-mouse IL-6R antibody (MR16-1). It is likely that osteoblasts and osteoclast progenitors are capable of transducing a signal from a complex of IL-6 and sIL-6R through gp130, even though they may have no or a very small number of IL-6Rs. Factors such as IL-11, oncostatin M, and leukemia inhibitory factor, which are known to exert their functions through gp130 (the signal-transducing chain of IL-6R), also induced MNC formation in our coculture system. These results suggest that increased circulating or locally produced sIL-6R induces osteoclast formation in the presence of IL-6 mediated by a mechanism involving gp130. This may play an important physiological or pathological role in conditions associated with increased osteoclastic bone resorption.
Mathematical textbook of deformable neuroanatomies.
Authors: Miller M I , Christensen G E , Amit Y , Grenander U
Published: May 2006

Mathematical techniques are presented for the transformation of digital anatomical textbooks from the ideal to the individual, allowing for the representation of the variabilities manifest in normal human anatomies. The ideal textbook is constructed on a fixed coordinate system to contain all of the information currently available about the physical properties of neuroanatomies. This information is obtained via sensor probes such as magnetic resonance, as well as computed axial and emission tomography, along with symbolic information such as white- and gray-matter tracts, nuclei, etc. Human variability associated with individuals is accommodated by defining probabilistic transformations on the textbook coordinate system, the transformations forming mathematical translation groups of high dimension. The ideal is applied to the individual patient by finding the transformation which is consistent with physical properties of deformable elastic solids and which brings the coordinate system of the textbook to that of the patient. Registration, segmentation, and fusion all result automatically because the textbook carries symbolic values as well as multisensor features.
Transcriptional regulatory elements downstream of the JunB gene.
Authors: Perez-Albuerne E D , Schatteman G , Sanders L K , Nathans D
Published: May 2006

JunB is an immediate early transcription factor that is induced by a variety of extracellular signaling agents, including growth factors, phorbol esters, and agents that elevate cyclic AMP. The mechanism of activation of the gene encoding JunB by these agents is not well understood. By using the JunB gene together with flanking DNA in transfection experiments, we show that a serum response element (SRE) and/or a cAMP response element (CRE) downstream of the gene mediate the response of the gene in mouse NIH 3T3 cells to serum, platelet-derived growth factor, basic fibroblast growth factor, phorbol ester, and forskolin. In addition, a segment of DNA just upstream of the TATA box is required for optimal activation of the gene.
Single photon emission computed tomographic imaging demonstrates loss of striatal dopamine transporters in Parkinson disease.
Authors: Innis R B , Seibyl J P , Scanley B E , Laruelle M , Abi-Dargham A , Wallace E , Baldwin R M , Zea-Ponce Y , Zoghbi S , Wang S
Published: May 2006

[123I][(1R)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane] ([123I]beta-CIT) labels dopamine transporters and is, therefore, a marker of neurons that degenerate in Parkinson disease. Single photon emission computed tomography imaging with [123I]beta-CIT showed that radioactivity in striatal regions in healthy subjects increased during a 2-day imaging study, whereas that in Parkinsonian patients peaked earlier at reduced levels relative to healthy subjects. Kinetic analyses of radioactivity in plasma and brain suggest that this decrease was due to an approximately 65% loss of target sites in patients compared with healthy subjects; greater losses occurred in putamen than in caudate. All patients showed lateralized differences in striatal uptake, with greater losses in the striatum contralateral to the side of the body with initial symptoms. These preliminary results suggest that [123I]beta-CIT is a marker for the loss of striatal dopamine terminals in patients with Parkinson disease. Single photon emission computed tomographic imaging with [123I]beta-CIT may be useful for early diagnosis of the disorder, for monitoring the progression of the disease, and for distinguishing the idiopathic disorder from other Parkinsonian syndromes with more widespread pathology.
Number of CpG islands and genes in human and mouse.
Authors: Antequera F , Bird A
Published: May 2006

Estimation of gene number in mammals is difficult due to the high proportion of noncoding DNA within the nucleus. In this study, we provide a direct measurement of the number of genes in human and mouse. We have taken advantage of the fact that many mammalian genes are associated with CpG islands whose distinctive properties allow their physical separation from bulk DNA. Our results suggest that there are approximately 45,000 CpG islands per haploid genome in humans and 37,000 in the mouse. Sequence comparison confirms that about 20% of the human CpG islands are absent from the homologous mouse genes. Analysis of a selection of genes suggests that both human and mouse are losing CpG islands over evolutionary time due to de novo methylation in the germ line followed by CpG loss through mutation. This process appears to be more rapid in rodents. Combining the number of CpG islands with the proportion of island-associated genes, we estimate that the total number of genes per haploid genome is approximately 80,000 in both organisms.
The gene responsible for Werner syndrome may be a cell division "counting" gene.
Authors: Faragher R G , Kill I R , Hunter J A , Pope F M , Tannock C , Shall S
Published: May 2006

Werner syndrome is a rare, autosomal, recessive condition that is frequently studied as a model of some aspects of human aging, although the behavioral changes that are usually associated with old age are only seen very infrequently. A most striking aspect of the phenotype of Werner syndrome, presumably arising from the same gene defect, is a dramatic shortening of the replicative life-span of dermal fibroblasts in vitro. The finite replicative life-span of human cells in vitro is due to the stochastic loss of replicative ability in a continuously increasing fraction of newborn cells at every generation. Normal human fibroblasts achieve approximately 60 population doublings in culture, while Werner syndrome cells usually only achieve approximately 20 population doublings. We describe an analysis of the replicative ability of fibroblasts from Werner syndrome patients and demonstrate that the cells in these cultures usually exit, apparently irreversibly, from the cell cycle at a faster rate than do normal cells, although they mostly start off with a good replicative ability. We propose that the Werner syndrome gene is a "counting" gene controlling the number of times that human cells are able to divide before terminal differentiation.
Protein kinase C and F1/GAP-43 gene expression in hippocampus inversely related to synaptic enhancement lasting 3 days.
Authors: Meberg P J , Barnes C A , McNaughton B L , Routtenberg A
Published: May 2006

The mRNA levels of protein F1 (also known as GAP-43), and protein kinase C (PKC) subtypes were measured 3 days after the induction of long-term enhancement (also known as long-term potentiation) in the hippocampus of chronically prepared conscious rats by quantitative in situ hybridization. Altered mRNA levels correlated significantly with alternations in synaptic efficacy; such correlations have not been reported previously. Rats with greater synaptic enhancement had lower gene expression in the CA3 subfield of F1/GAP-43 and both beta-PKC and gamma-PKC, but not alpha-PKC. For microtubule-associated protein 2 (MAP-2), neurogranin, and the glutamate receptor subtype B-flip, no correlation was observed in any cell field between synaptic enhancement and hybridization to the mRNA. To our surprise, alterations in mRNA levels of F1/GAP-43 and gamma-PKC were highly correlated (r = +0.928, P < 0.001), suggesting coordinate regulation. Since F1/GAP-43 is associated with neurite growth, its lowered expression at 3 days would reduce potential growth, leading to synaptic stabilization. We propose that long-term synaptic change is mediated by gene expression of the very same proteins initially modified posttranslationally.
Benzo[a]pyrene-induced murine skin tumors exhibit frequent and characteristic G to T mutations in the p53 gene.
Authors: Ruggeri B , DiRado M , Zhang S Y , Bauer B , Goodrow T , Klein-Szanto A J
Published: May 2006

Human tobacco-related cancers exhibit a high frequency of G to T transversions in the mutation hot spot region of the p53 tumor suppressor gene, possibly the result of specific mutagens in tobacco smoke, most notably benzo[a]pyrene (B[a]P). No in vivo animal model of B[a]P-induced tumorigenesis has been used, however, to substantiate these molecular epidemiological data experimentally. Direct DNA sequence analysis of the hot spot region (exons 5-8 inclusive) of murine p53 was performed in 20 skin tumors induced by a complete carcinogenesis protocol with B[a]P. Sequence analyses revealed numerous heterozygous missense mutations in carcinomas, specifically in exons 7 and 8 of the p53 gene, and targeting exclusively guanine residues. Moreover, 70% (5/7) of the mutations characterized were G to T transversions. In contrast, direct DNA sequence analysis of 36 skin tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in either a complete carcinogenesis protocol or in a two-stage carcinogenesis protocol revealed a 30% frequency of heterozygous p53 mutations, with the majority of mutations found in carcinomas, but only a single G to T transversion (1/8). Thus, while mutation frequencies are similar, the pattern and type of p53 mutations in B[a]P-induced skin tumors differs significantly from the mutation spectra in DMBA-induced squamous neoplasias. These in vivo findings in B[a]P-induced tumors lend support to in vitro and molecular epidemiological evidence, suggesting that the p53 tumor suppressor gene may be a selective target of metabolically activated B[a]P species etiologically associated with human tobacco-related cancers.
Mechanism of the cardioprotective effect of transforming growth factor beta 1 in feline myocardial ischemia and reperfusion.
Authors: Lefer A M , Ma X L , Weyrich A S , Scalia R
Published: May 2006

We studied the effects of transforming growth factor beta 1 (TGF-beta 1) in a feline model of myocardial ischemia (1.5 hr) and reperfusion (4.5 hr). Myocardial ischemia followed by reperfusion resulted in severe myocardial injury, endothelial dysfunction, high cardiac myeloperoxidase activity indicative of neutrophil accumulation in the ischemic myocardium, and significant neutrophil adherence to the ischemic coronary endothelium. In contrast, intravenous administration of TGF-beta 1 (20 micrograms/kg) 30 min prior to reperfusion significantly attenuated myocardial necrosis (13.8% +/- 3.5% vs. 32.2% +/- 2.9% of area-at-risk, P < 0.01) and attenuated endothelial dysfunction (P < 0.01) associated with ischemia-reperfusion. Moreover, myeloperoxidase activity in the ischemic myocardium was significantly lower than vehicle controls (0.2 +/- 0.1 vs. 1.7 +/- 0.3 units/100 mg of tissue, P < 0.01) and neutrophil adherence to ischemic coronary endothelium was significantly (P < 0.01) attenuated in TGF-beta 1-treated cats. These results demonstrate that TGF-beta 1 exerts a significant cardioprotective effect in a feline model of myocardial ischemia and reperfusion. The mechanism of this protective effect appears to relate to endothelial preservation by TGF-beta 1 inhibiting circulating neutrophils from adhering to the endothelium, a critical step in neutrophil-induced reperfusion injury.
Redundant cyclin overexpression and gene amplification in breast cancer cells.
Authors: Keyomarsi K , Pardee A B
Published: May 2006

Cyclins are prime cell cycle regulators and are central to the control of major check points in eukaryotic cells. The aberrant expressions of two cyclins (i.e., cyclins A and D1) have been observed in some cancers, suggesting they may be involved in loss of growth control. However, in spite of these occasional changes involving only two cyclins, there are no clear connections between general derangements of other cyclins or their dependent kinases in a single tumor type. We detected general cyclin overexpression in 3 of 3 breast tumor tissue samples. In addition, using proliferating normal vs. human tumor breast cells as a model system, we observed a number of alterations in cyclin expression: (i) an 8-fold amplification of cyclin E gene in one tumor line, a 64-fold overexpression of its mRNA, and altered expression of its protein; (ii) deranged expression of cyclin E protein in all (10 of 10) tumor cell lines studied; (iii) increased cyclin mRNA stability, resulting in (iv) general overexpression of RNAs and proteins for cyclins A and B and CDC2 in 9 of 10 tumor lines and (v) deranged order of appearance of cyclins in synchronized tumor vs. normal cells, with mitotic cyclins appearing prior to G1 cyclins. These multiple general derangements in cyclin expression in human breast cancer cells provide evidence linking aberrant cyclin expression to tumorigenesis.
Twist and writhe of a DNA loop containing intrinsic bends.
Authors: Bauer W R , Lund R A , White J H
Published: May 2006

The finite-element method of solid mechanics is applied to calculation of the three-dimensional structure of closed circular DNA, modeled as an elastic rod subject to large motions. The results predict the minimum elastic energy conformation of a closed loop of DNA as a function of relaxed equilibrium configuration and linking number (Lk). We apply the method to four different starting states: a straight rod, two rods containing either one or two 20 degrees bends, and a circular O-ring. The results, here at low superhelix density, show the changes in writhe (Wr) and in twist (Tw) as Lk is progressively lowered. The presence of even a single intrinsic bend reduces significantly the linking number change at which Wr first appears, compared to an initially straight, bend-free rod. The presence of two in-phase bends, situated at opposite ends of a diameter, leads to the formation of at least two distinct regions of different but relatively uniform Tw increment. The O-ring begins to writhe immediately upon reduction of Lk, and the Tw increment distribution is sinusoidal along the rod. The mechanics calculations, unlike other theoretical approaches, permit us to calculate Tw and Wr independent of the constraint of constant Lk.
Functional activation of the human frontal cortex during the performance of verbal working memory tasks.
Authors: Petrides M , Alivisatos B , Meyer E , Evans A C
Published: May 2006

Regional cerebral blood flow was measured with positron emission tomography during the performance of verbal working memory tasks. The same type of verbal response (i.e., reciting numbers) was required in the control and the two experimental tasks. In the control task, the subjects were required to count aloud. In the two experimental tasks, the subjects were required to maintain within working memory the numbers they generated (self-ordered task) or the numbers generated by the experimenter (externally ordered task). Examination of the difference in activation between these conditions revealed strong bilateral activation within the mid-dorsolateral frontal cortex during both experimental tasks. There was, however, no evidence of additional activation within the mid-dorsolateral frontal cortex when monitoring self-generated responses as compared with the monitoring of externally generated responses. These results provide evidence regarding the role of the mid-dorsolateral frontal cortex in mnemonic processing that are in agreement with recent findings from work with non-human primates.
Tumor-suppressive effect of the retinoic acid receptor beta in human epidermoid lung cancer cells.
Authors: Houle B , Rochette-Egly C , Bradley W E
Published: May 2006

Retinoic acid receptor beta (RAR beta), which codes for a nuclear receptor for retinoic acid, is localized in a chromosomal region frequently deleted in lung cancer cells. The gene is expressed in normal lung tissue and in the majority of the cell lines derived from lung tumors but not in most of the lines derived from lung tumors with epidermoid characteristics. To study the possible role of RAR beta in growth control of epidermoid lung tumor-derived cells, transfectants expresing RAR beta were generated from nonexpressing epidermoid tumor-derived cell lines. Four clones were derived from line CALU-1, three of which showed a 20-60% increase in doubling time in the presence of retinoic acid. Parental and control-transfected cells were unaffected or slightly stimulated. All four clones expressing RAR beta were less tumorigenic in nude mice than were the untransfected or control-transfected cells, with about a 50% incidence of take vs. 95%. When tumors did develop from RAR beta-positive cells, they showed a reduced rate of growth, an increased latency, and, in six of seven tumors tested, a much reduced level of RAR beta expression. Transfectants derived from a second tumor line, H157, also showed a markedly reduced incidence of take in nude mice. Together with the known effects of retinoic acid on differentiation and carcinogenesis, our results support the hypothesis that RAR beta functions as a tumor suppressor gene in epidermoid lung tumorigenesis.
Identification of C-terminal amino acid residues of cauliflower mosaic virus open reading frame III protein responsible for its DNA binding activity.
Authors: Mougeot J L , Guidasci T , Wurch T , Lebeurier G , Mesnard J M
Published: May 2006

We cloned in Escherichia coli truncated versions of the protein p15 encoded by open reading frame III of cauliflower mosaic virus. We then compared the ability of the wild-type p15 (129 amino acids) and the deleted p15 to bind viral double-stranded DNA genome. Deletions of > 11 amino acids in the C-terminal proline-rich region resulted in loss of DNA binding activity of wild-type p15. Moreover, a point mutation of the proline at position 118 sharply reduced the interaction between the viral protein and DNA. These results suggest that cauliflower mosaic virus p15 belongs to the family of DNA binding proteins having a proline-rich motif involved in interaction with double-stranded DNA.
The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation.
Authors: Benning C , Beatty J T , Prince R C , Somerville C R
Published: May 2006

All photosynthetic organisms, with the exception of several species of photosynthetic bacteria, are thought to contain the sulfolipid 6-sulfo-alpha-D-quinovosyldiacylglycerol. The association of this lipid with photosynthetic membranes has led to the assumption that it plays some role in photosynthesis. Stable null mutants of the photosynthetic bacterium Rhodobacter sphaeroides completely lacking sulfolipid were obtained by disruption of the sqdB gene. The ratios of the various components of the photosynthetic electron transport chain, as well as the electron transfer rates during cyclic electron transport, were not altered in the mutants, when grown under optimal conditions. Growth rates of wild type and mutants were identical under a variety of growth conditions, with the exception of phosphate limitation, which resulted in reduced growth of the mutants. Phosphate limitation of the wild type caused a significant reduction in the amount of all phospholipids and an increased amount of sulfolipid. By contrast, the sulfolipid-deficient mutant had reduced levels of phosphatidylcholine and phosphatidylethanolamine but maintained a normal level of phosphatidylglycerol. In addition, two unidentified lipids lacking phosphorus accumulated in the membranes of both wild-type and mutant strains under phosphate limitation. We conclude that sulfolipid plays no significant unique role in photoheterotrophic growth or photosynthetic electron transport in R. sphaeroides but may function as a surrogate for phospholipids, particularly phosphatidylglycerol, under phosphate-limiting conditions.
DNA unwinding upon strand-displacement binding of a thymine-substituted polyamide to double-stranded DNA.
Authors: Cherny D Y , Belotserkovskii B P , Frank-Kamenetskii M D , Egholm M , Buchardt O , Berg R H , Nielsen P E
Published: May 2006

It was recently found that polyamide nucleic acid (PNA) analogues consisting of thymines attached to an aminoethylglycine backbone bind strongly and sequence-selectively to adenine sequences of oligonucleotides and double-stranded DNA [Nielsen, P. E., Egholm, M., Berg, R. H. & Buchardt, O. (1991) Science 254, 1497-1500]. It was concluded that the binding to double-stranded DNA was accomplished via strand displacement, in which the PNA bound to the Watson-Crick complementary adenine-containing strand, whereas the thymine-containing strand was extruded in a virtually single-stranded conformation. This model may provide a general way in which to obtain sequence-specific recognition of any sequence in double-stranded DNA by Watson-Crick hydrogen-bonding base-pair recognition, and it is thus paramount to rigorously establish this binding mode for synthetic DNA-binding ligands. We now report such results from electron microscopy. Furthermore, we show that binding of PNA to closed circular DNA results in unwinding of the double helix corresponding to approximately one turn of the double helix per 10 base pairs. The DNA.PNA complex, which is formed at low salt concentration (only a small portion of DNA molecules show complex formation at NaCl concentration higher than 40 mM), is exceptionally kinetically stable and cannot be dissociated by increasing salt concentration up to 500 mM.
Potent and highly selective human immunodeficiency virus type 1 (HIV-1) inhibition by a series of alpha-anilinophenylacetamide derivatives targeted at HIV-1 reverse transcriptase.
Authors: Pauwels R , Andries K , Debyser Z , Van Daele P , Schols D , Stoffels P , De Vreese K , Woestenborghs R , Vandamme A M , Janssen C G
Published: May 2006

In vitro evaluation of a large chemical library of pharmacologically acceptable prototype compounds in a high-capacity, cellular-based screening system has led to the discovery of another family of human immunodeficiency virus type 1 (HIV-1) inhibitors. Through optimization of a lead compound, several alpha-anilinophenylacetamide (alpha-APA) derivatives have been identified that inhibit the replication of several HIV-1 strains (IIIB/LAI, RF, NDK, MN, HE) in a variety of host cell types at concentrations that are 10,000- to 100,000-fold lower than their cytotoxic concentrations. The IC50 of the alpha-APA derivative R 89439 for HIV-1 cytopathicity in MT-4 cells was 13 nM. The median 90% inhibitory concentration (IC90) in a variety of host cells was 50-100 nM. Although these alpha-APA derivatives are active against a tetrahydroimidazo [4,5,1-jk][1,4]benzodiazepin-2(1H)-thione-(TIBO)-resistant HIV-1 strain, they do not inhibit replication of HIV-2 (strains ROD and EHO) or simian immunodeficiency virus (strains Mac251, mndGB1, and agm3). An HIV-1 strain containing the Tyr181-->Cys mutation in the reverse transcriptase region displayed reduced sensitivity. alpha-APA derivative R 89439 inhibited virion and recombinant reverse transcriptase of HIV-1 but did not inhibit that of HIV-2. Reverse transcriptase inhibition depended upon the template/primer used. The relatively uncomplicated synthesis of R 89439, its potent anti-HIV-1 activity, and its favorable pharmacokinetic profile make R 89439 a good candidate for clinical studies.
Activation of the c-Ki-ras oncogene in aflatoxin B1-induced hepatocellular carcinoma and adenoma in the rat: detection by denaturing gradient gel electrophoresis.
Authors: Soman N R , Wogan G N
Published: May 2006

Sequence alterations in the exon 1 region of the rat c-Ki-ras gene were studied in DNA isolated from aflatoxin B1 (AFB1)-induced rat liver carcinomas and precursor lesions appearing 56 weeks after administration of the carcinogen. To detect the mutations with high sensitivity, DNA samples were analyzed by using polymerase chain reaction (PCR) amplification in conjunction with allele-specific oligonucleotide (ASO) hybridization together with a modified PCR-G+C clamp-denaturing gradient gel electrophoresis (DGGE) method. Mutations in the Ki-ras gene were present in all adenomas and carcinomas examined. The predominant mutation observed was a G.C-to-A.T base transition in codon 12 (GGT to GAT). Also present, but at low frequency, was a G.C-to-T.A base transversion in the same codon (GGT to TGT). In addition, 20% of the samples contained a G.C-to-T.A transversion in the second base position of codon 12 (GGT to GTT), a mutation not previously observed in AFB1-induced rat liver tumors. These results confirm and extend our previous findings that Ki-ras mutation is a prevalent event in hepato-cellular carcinogenesis induced in Fischer 344 rats by AFB1. The modified DGGE method described is applicable to the screening of multiple mutations in neoplastic lesions with high fidelity and sensitivity.
Allosteric transition of fructose-1,6-bisphosphatase.
Authors: Liang J Y , Zhang Y , Huang S , Lipscomb W N
Published: May 2006

Structural changes during the R-to-T transition of fructose-1,6-bisphosphatase (EC 3.1.3.11) form a hierarchy, in which structural changes at one level are supported by those at the other levels. The quaternary conformational changes involve a 17 degrees rotation between the upper and lower dimers, and a 3.4 degrees rotation between monomers in a dimer. Within monomers, the FBP domain, which remains rigid during the R-to-T transition, rotates 2.3 degrees relative to the AMP domain, which undergoes significant structural reorientations. The most important of these reorientations are the newly identified partially ordered loop residues 55-61 in the T state and reorientations of helices H1, H2, and H3. Supporting these structural changes are numerous readjustments of hydrogen bonding and van der Waals interactions throughout the entire tetrameric protein. Propagation of structural changes during the R-to-T transition relies primarily on helices H1, H2, H3, and loop 50-72. The change that begins at the AMP site causes reorientation of H1, H2, and H3 and changes of interactions across the C1-C4 (C2-C3) interface. These changes may propagate down H1, H2, H3, and loop 50-72 to affect interactions across the C1-C2 (C3-C4) and C1-C3 (C2-C4) interfaces. AMP inhibition is most probably caused by reduced metal binding affinity due to structural changes of metal ligands (Glu97, Asp118, and Asp121) in the active site. The eight-stranded beta-sheet, particularly the beta-strand B3, which connects Lys112 and Tyr113 of the AMP site with Asp118 and Asp121 of the metal site, may be responsible for communication between the AMP and active sites. Additional structural changes that support such communication include reorientation of the FBP domain and H1, H2, and H3 relative to the eight-stranded beta-sheet, and new conformations of loop 54-72 in the T state as AMP binds.
Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiency with normal or elevated IgM.
Authors: Fuleihan R , Ramesh N , Loh R , Jabara H , Rosen R S , Chatila T , Fu S M , Stamenkovic I , Geha R S
Published: May 2006

B lymphocytes from patients with X chromosome-linked immunoglobulin deficiency with normal or elevated serum IgM are unable to switch from the synthesis of IgM/IgD to that of other immunoglobulin isotypes. Isotype switch recombination was evaluated in three affected males by examining interleukin 4-driven IgE synthesis. T-cell-dependent IgE synthesis was completely absent in the B lymphocytes of the patients. In contrast, CD40 mAb plus interleukin 4 induced the patients' B cells to synthesize IgE and to undergo deletional switch recombination. Because interaction between CD40 and its ligand on activated T cells is critical for T-cell-driven isotype switching, we examined CD40 ligand expression. In contrast to normal T cells, lymphocytes from the patients expressed no detectable CD40 ligand on their surface after stimulation with phorbol 12-myristate 13-acetate and ionomycin, although the mRNA of the ligand was expressed normally. These results suggest that defective expression of the CD40 ligand underlies the failure of isotype switching in this disease.
Different AIDS incubation periods and their impacts on reconstructing human immunodeficiency virus epidemics and projecting AIDS incidence.
Authors: Bacchetti P , Segal M R , Hessol N A , Jewell N P
Published: May 2006

The objective of this study was to investigate heterogeneity in incubation distributions in different cohorts and to assess the sensitivity of back-calculated infection rates to different assumptions about incubation times from human immunodeficiency virus (HIV) infection to AIDS diagnosis. Incubation distributions were estimated by using data from three different cohort studies. These and one other published incubation model were used as inputs for a back-calculation procedure that reconstructed smooth HIV-infection rates from AIDS incidence among adults in the United States, allowing for changes over time in incubation. Incubation estimates from the different cohorts differed substantially. The cumulative HIV incidence estimates that result from using the different incubations are very different, but the back-calculated models all produce good fits to the observed diagnosis counts. We conclude that systematic differences in incubation times of different groups add substantially to the uncertainty inherent in using the back-calculation method to reconstruct HIV epidemics and project future numbers of AIDS cases.
Existence of an inactive pool of acetylcholinesterase in chicken brain.
Authors: Chatel J M , Grassi J , Frobert Y , Massoulié J , Vallette F M
Published: May 2006

We analyzed acetylcholinesterase (AcChoEase; EC 3.1.1.7) activity and AcChoEase immunoreactive protein in chicken brain by using five monoclonal antibodies raised against chicken AcChoEase. Four of them specifically recognized AcChoEase catalytic subunits in Western blots and one, C-131, recognized only enzymatically active AcChoEase. We observed considerable differences in the ratio of immunoreactive protein to catalytic activity in various fractions, indicating the existence of inactive AcChoEase protein. This inactive AcChoEase component was more abundant in a low-salt-soluble extract than in a subsequent detergent-soluble extract. On the basis of the ratio between activity and immunoreactivity, we calculated that the inactive component represents about 30% of the total AcChoEase subunits in chicken brain. The immunoreactive AcChoEase protein sedimented in sucrose gradients like the active molecular forms; the G1 and G2 peaks contained inactive molecules, whereas the G4 peak appeared to contain only active AcChoEase. The bulk of inactive AcChoEase reacted with the organophosphate cholinesterase inhibitor O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonothioate (MTP) but was found to bind the active site affinity ligand N-methylacridinium poorly and was not recognized by the active-form-specific monoclonal antibody, C-131. In addition, most of this fraction is sensitive to endoglycosidase H and binds the lectin wheat germ agglutinin poorly, suggesting that it was not processed in the Golgi apparatus. From these observations, we propose that the active and inactive AcChoEase components are differently folded.
A murine retrovirus induces proliferation of unique lymphoid cell lines expressing T-cell-receptor structures utilizing common variable region alpha and beta chain genes.
Authors: O'Neill H C
Published: May 2006

A murine radiation leukemia virus (RadLV) has been shown to induce in vitro proliferation of an unusual subset of lymphoid cells from spleen. They have the unusual property of expressing CD3/T-cell receptor alpha and beta chains (TCR-alpha beta) in the absence of other T-cell markers such as Thy-1, CD4, and CD8. Cell lines induced in two mouse strains with RadLV produced by the C6VL/1 thymoma all specifically utilize common V alpha 3 and V beta 8.2 variable region genes in the formation of a TCR structure. Each of these cell lines has now been found to express both class I and class II major histocompatibility antigens and the beta 2 integrin specific for spleen dendritic cells. Analysis of functional properties of these cells has revealed a subset that can endocytose proteins and function alone as antigen-presenting cells. They have therefore been called D-T cells (i.e., dendritic cells expressing TCR-alpha beta). A retroviral-driven event has been implicated in the proliferation of dendritic-like cells expressing a common TCR epitope.
Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor.
Authors: Fantini J , Cook D G , Nathanson N , Spitalnik S L , Gonzalez-Scarano F
Published: May 2006

The gastrointestinal tract plays a major role in the pathogenesis and pathophysiology of infection by the type 1 human immunodeficiency virus (HIV-1). It is a potential route for viral entry and it is the site of a number of complications, including both opportunistic infections and a primary HIV-induced enteropathy. Correspondingly, both in vivo and in vitro studies have demonstrated HIV infection of gastrointestinal cells of lymphoid and epithelial origin. HT-29, a human colonic epithelial cell line that is infectable with many HIV-1 strains, does not express CD4 protein or mRNA. Recent studies showed that antibodies recognizing a neutral glycolipid related to galactosylceramide (GalCer) in HT-29 cells inhibited HIV-1 infection of this cell line, extending previous findings in neural cells. In the current studies, we further analyzed the neutral glycolipids of HT-29 cells and showed that they contained authentic GalCer and that recombinant gp120 bound to this glycolipid. Moreover, by analyzing GalCer expression in clones derived from HT-29 and Caco-2 (another human colonic cell line), we observed that the level of expression of this glycolipid was associated with the sensitivity to HIV-1 infection. Subclones of Caco-2 did not express GalCer and were not infectable with any of three HIV-1 strains. These results strengthen the possibility that GalCer is an alternative receptor in CD4- cell lines. Furthermore, since GalCer is a major glycolipid in epithelial cells of the small intestine and colon, these results provide a structural basis for the binding of HIV-1 by gastrointestinal epithelial cells and the entry of the virus into those cells.
Isolation of interleukin 2-induced immediate-early genes.
Authors: Beadling C , Johnson K W , Smith K A
Published: May 2006

Clonal expansion of antigen-reactive T lymphocytes is driven by the lymphokine interleukin 2 (IL-2). To further elucidate the mechanisms of IL-2 action, we have utilized a differential hybridization procedure to clone IL-2-induced immediate-early genes from an IL-2-stimulated human T-cell cDNA library. To increase the frequency of IL-2-induced transcripts represented in the library, the protein synthesis inhibitor cycloheximide was included during the 2-hr IL-2 stimulation to superinduce gene expression, and the uridine analogue 4-thiouridine was utilized to enable selective purification of newly synthesized transcripts. From the enriched library, we have isolated eight IL-2-induced genes, six of which represent previously unrecognized human sequences. Northern blot analysis revealed that the induction of seven of the genes is specific to the IL-2-mediated G1 "progression" phase of the cell cycle, in that only one gene is also induced during the T-cell receptor-triggered G0-G1 "competence" phase. These results indicate that the effects of IL-2 are mediated by the specific induction of a number of immediate-early genes and provide a means with which to further delineate the mechanisms whereby IL-2 stimulates T-lymphocyte proliferation and differentiation. The methods described in this report should also be of general utility in the dissection of the signaling pathways activated by diverse cytokine receptors.
Cloning of the cDNA encoding a myosin heavy chain B isoform of Xenopus nonmuscle myosin with an insert in the head region.
Authors: Bhatia-Dey N , Adelstein R S , Dawid I B
Published: May 2006

The complete amino acid sequence of Xenopus laevis nonmuscle myosin heavy chain B (MHC-B) has been deduced from overlapping cDNA clones isolated from an XTC cell library. RNA blots of various developmental stages, adult tissues, and XTC cells detect a single transcript of 7.5 kb which is expressed at similar levels throughout development. MHC-B mRNA was detected in XTC cells, heart, lung, spleen, and brain, at lower levels in ovary, testis, pancreas, stomach, liver, and eye, but not in kidney and skeletal muscle. Protein expression in adult tissues, as detected by immunoblot analysis, correlates well with mRNA expression. In chickens and humans, a fraction of the mRNA encoding the MHC-B isoform was found previously to contain a 10-amino acid insert at amino acid 211 near the ATP-binding site. As reported elsewhere, in the chicken this insert-bearing isoform is nervous system-specific. The Xenopus sequence shows a 16-amino acid insertion at the same position; 7 of 16 residues are identical to those in the chicken and human insertion, and these identical residues include a consensus target sequence for cyclin-p34cdc2 kinase. In contrast to chicken, all frog tissues and embryonic stages tested contained the insert-bearing form, and no evidence for a non-insert-bearing MHC-B isoform was found in Xenopus.