bluuebunny/crossref_metadata_2025 huggingface dataset:
Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance.
Authors: Delaney T P , Friedrich L , Ryals J A
Published: May 2006

Plants possess multiple resistance mechanisms that guard against pathogen attack. Among these are inducible systems such as systemic acquired resistance (SAR). SAR is activated by pathogen exposure and leads to an increase in salicylic acid (SA), high-level expression of SAR-related genes, and resistance to a spectrum of pathogens. To identify components of the signal transduction pathways regulating SAR, a mutant screen was developed that uses 2,6-dichloroisonicotinic acid as an activator of SAR gene expression and pathogen resistance, followed by assays for resistance to the fungal pathogen Peronospora parasitica. Mutants from this screen were subsequently examined to assess their defense responses. We describe here a recessive mutation that causes a phenotype of insensitivity to chemical and biological inducers of SAR genes and resistance. These data indicate the existence of a common signaling pathway that couples these diverse stimuli to induction of SAR genes and resistance. Because of its non-inducible immunity phenotype, we call this mutant nim1. Although nim1 plants fail to respond to SA, they retain the ability to accumulate wild-type levels of SA, a probable endogenous signal for SAR. Further, the ability of nim1 plants to support growth of normally incompatible races of a fungal pathogen indicates a role for this pathway in expression of genetically determined resistance, consistent with earlier findings for transgenic plants engineered to break down SA. These results suggest that the wild-type NIM1 gene product functions in a pathway regulating acquired resistance, at a position downstream of SA accumulation and upstream of SAR gene induction and expression of resistance.
cAMP-dependent, long-lasting inhibition of a K+ current in mammalian neurons.
Authors: Ansanay H , Dumuis A , Sebben M , Bockaert J , Fagni L
Published: May 2006

We report the long-term modulation of K+ channels by cAMP in cultured murine colliculi neurons. A short (1-2 s) application of 8-Br-cAMP induced a long-lasting broadening of the action potential, a loss of after-hyperpolarization, and a reduction in spike accommodation. In agreement with these changes, 8-Br-cAMP produced a long-lasting (2 hr) inhibition of a K+ current. These effects were also observed after a short activation of the pituitary adenylyl cyclase-activating polypeptide, beta-adrenergic, and 5-hydroxytryptamine type 4 (5-HT4) receptors, all known to increase cAMP. A transient activation of the cAMP-dependent protein kinase and a long-lasting inhibition of phosphatases (up to 2 hr) were detected. The blockade of the K+ current resulting from a brief application of 8-Br-cAMP or 5-hydroxytryptamine was prolonged from 2 to 4 hr when protein-serine/threonine phosphatases 1 and 2A were inhibited with 10 nM okadaic acid. The critical steps following the cAMP-dependent protein kinase activation and resulting in a long-term blockade of phosphatases are discussed in this report.
The physics and chemistry of heavy fermions.
Authors: Fisk Z , Sarrao J L , Smith J L , Thompson J D
Published: May 2006

The heavy fermions are a subset of the f-electron intermetallic compounds straddling the magnetic/nonmagnetic boundary. Their low-temperature properties are characterized by an electronic energy scale of order 1-10 K. Among the low-temperature ground states observed in heavy fermion compounds are exotic superconductors and magnets, as well as unusual semiconductors. We review here the current experimental and theoretical understanding of these systems.
Rescue of defective mitogenic signaling by D-type cyclins.
Authors: Roussel M F , Theodoras A M , Pagano M , Sherr C J
Published: May 2006

Three gene products, including Myc and the D- and E-type G1 cyclins, are rate limiting for G1 progression in mammalian fibroblasts. Quiescent mouse NIH 3T3 fibroblasts engineered to express a mutant colony-stimulating factor (CSF-1) receptor (CSF-1R 809F) fail to synthesize c-myc and cyclin D1 mRNAs upon CSF-1 stimulation and remain arrested in early G1 phase. Ectopic expression of c-myc or either of three D-type cyclin genes, but not cyclin E, resensitized these cells to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. Rescue by cyclin D1 was enhanced by c-myc but not by cyclin E and was reversed by infecting cyclin D1-reconstituted cells with a retroviral vector encoding catalytically inactive cyclin-dependent kinase 4. Induction of cyclin D1 mRNA by CSF-1 was restored in cells forced to express c-myc, and vice versa, suggesting that expression of the two genes is interdependent. Cells reconstituted with c-myc were prevented from entering S phase when microinjected with a monoclonal antibody to cyclin D1, and conversely, those rescued by cyclin D1 were inhibited from forming CSF-1-dependent colonies when challenged with a dominant-negative c-myc mutant. Cyclin D mutants defective in binding to the retinoblastoma protein were impaired in rescuing mitogenic signaling. Therefore, Myc and D-type cyclins collaborate during the mitogenic response to CSF-1, whereas cyclin E functions in a separate pathway.
Interleukin 12 suppresses autoantibody production by reversing helper T-cell phenotype in hepatitis B e antigen transgenic mice.
Authors: Milich D R , Wolf S F , Hughes J L , Jones J E
Published: May 2006

Helper T (Th) cells are classified as Th1 or Th2 cells by virtue of cytokine secretion and function as mediators of cellular or humoral immunity, respectively. Cytokines also regulate the differentiation of Th cells. For example, interleukin (IL)-12 promotes Th1 and suppresses Th2 cell development, suggesting that IL-12 may be useful therapeutically in Th2-mediated autoimmune and allergic disorders. Therefore, the effect of systemic IL-12 treatment on in vivo autoantibody synthesis in hepatitis B e antigen (HBeAg)-expressing transgenic mice, which is dependent on self-reactive Th2 cells, was examined. Low-dose IL-12 significantly inhibited autoantibody production by shifting the Th2-mediated response toward Th1 predominance. Additionally, previous studies suggest that a predominance of HBeAg-specific Th2-type cells may contribute to chronicity in hepatitis B virus infection. Therefore, IL-12 may also prove beneficial in modulating the HBeAg-specific Th response to favor viral clearance in chronic hepatitis B virus infection.
On the existence and implications of an inverse folding code in proteins.
Authors: Rackovsky S
Published: May 2006

The existence of a code relating the set of possible sequences at a given position in a protein backbone to the local structure at that location is investigated. It is shown that only 73% of 4-C alpha structure fragments in a sample of 114 protein structures exhibit a preference for a particular set of sequences. The remaining structures can accommodate essentially any sequence. The structures that encode specific sequence distributions include the classical "secondary" structures, with the notable exception of planar (beta) bends. It is suggested that this has implications as to the mechanism of folding in proteins with extensive sheet/barrel structure. The possible role of structures that do not encode specific sequences as mutation hot spots is noted.
Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.
Authors: Wargnier A , Legros-Maida S , Bosselut R , Bourge J F , Lafaurie C , Ghysdael C J , Sasportes M , Paul P
Published: May 2006

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.
Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins.
Authors: Peterson D S , Miller L H , Wellems T E
Published: May 2006

Open reading frames in the Plasmodium falciparum genome encode domains homologous to the adhesive domains of the P. falciparum EBA-175 erythrocyte-binding protein (eba-175 gene product) and those of the Plasmodium vivax and Plasmodium knowlesi Duffy antigen-binding proteins. These domains are referred to as Duffy binding-like (DBL), after the receptor that determines P. vivax invasion of Duffy blood group-positive human erythrocytes. Using oligonucleotide primers derived from short regions of conserved sequence, we have developed a reverse transcription-PCR method that amplifies sequences encoding the DBL domains of expressed genes. Products of these reverse transcription-PCR amplifications include sequences of single-copy genes (including eba-175) and variably transcribed genes that cross-hybridize to multiple regions of the genome. Restriction patterns of the multicopy genes show a high degree of polymorphism among different parasite lines, whereas single-copy genes are generally conserved. Characterization of the single-copy genes has identified a gene (ebl-1) that is related to eba-175 and is likely to be involved in erythrocyte invasion.
Photocleavable biotin derivatives: a versatile approach for the isolation of biomolecules.
Authors: Olejnik J , Sonar S , Krzymañska-Olejnik E , Rothschild K J
Published: May 2006

While the strong biotin-avidin interaction has been widely used for the detection of biomolecules, its irreversibility complicates their isolation. We report the synthesis of a photocleavable biotin derivative (PCB) which eliminates many limitations of existing methods. This reagent contains a biotin moiety linked through a spacer arm to a photocleavable moiety, which reacts selectively with primary amino groups on any substrate. In experiments using [leucine]-enkephalin as a model substrate, we show that PCB retains its high affinity toward avidin/streptavidin and allows rapid (< 5 min) and efficient (> 99%) photorelease of the substrate in a completely unaltered form. Photocleavable biotins should be useful in numerous applications involving the isolation of proteins, nucleic acids, lipids, and cells.
Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer.
Authors: Li T , Davidson B L
Published: May 2006

We have studied the use of adenovirus-mediated gene transfer to reverse the pathologic changes of lysosomal storage disease caused by beta-glucuronidase deficiency in the eyes of mice with mucopolysaccharidosis VII. A recombinant adenovirus carrying the human beta-glucuronidase cDNA coding region under the control of a non-tissue-specific promoter was injected intravitreally or subretinally into the eyes of mice with mucopolysaccharidosis VII. At 1-3 weeks after injection, the treated and control eyes were examined histochemically for beta-glucuronidase expression and histologically for phenotypic correction of the lysosomal storage defect. Enzymatic expression was detected 1-3 weeks after injection. Storage vacuoles in the retinal pigment epithelium (RPE) were still present 1 week after gene transfer but were reduced to undetectable levels by 3 weeks in both intravitreally and subretinally injected eyes. There was minimal evidence of ocular pathology associated with the viral injection. These data indicate that adenovirus-mediated gene transfer to the eye may provide for adjunctive therapy for lysosomal storage diseases affecting the RPE in conjunction with enzyme replacement and/or gene therapies for correction of systemic disease manifestations. The data also support the view that recombinant adenovirus may be useful as a gene therapy vector for retinal degenerations that result from a primary genetic defect in the RPE cells.
Structural domains of IS10 transposase and reconstitution of transposition activity from proteolytic fragments lacking an interdomain linker.
Authors: Kwon D , Chalmers R M , Kleckner N
Published: May 2006

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.
Simian virus 40 late gene expression is regulated by members of the steroid/thyroid hormone receptor superfamily.
Authors: Zuo F , Mertz J E
Published: May 2006

Transcription of the late genes of simian virus 40 (SV40) is repressed during the early phase of the lytic cycle of infection of binding of cellular factors, called IBP-s, to the SV40 late promoter; repression is relieved after the onset of viral DNA replication by titration of these repressors. Preliminary data indicated that one of the major components of IBP-s was human estrogen-related receptor 1 (hERR1). We show here that several members of the steroid/thyroid hormone receptor superfamily, including testis receptor 2, thyroid receptor alpha 1 in combination with retinoid X receptor alpha, chicken ovalbumin upstream promoter transcription factors 1 and 2 (COUP-TF1 and COUP-TF2), as well as hERR1, possess the properties of IBP-s. These receptors bind specifically to hormone receptor binding sites present in the SV40 major late promoter. Recombinant COUP-TF1 specifically represses transcription from the SV40 major late promoter in a cell-free transcription system. Expression of COUP-TF1, COUP-TF2, or hERR1 in monkey cells results in repression of the SV40 late promoter, but not the early promoter, in the absence of the virally encoded large tumor antigen. Overexpression of COUP-TF1 leads to a delay in the early-to-late switch in SV40 gene expression during the lytic cycle of infection. Thus, members of this superfamily can play major direct roles in regulating expression of SV40. Possibly, natural or synthetic ligands to these receptors can serve as antiviral drugs. Our findings also provide the basis for the development of assays to screen for the ligands to testis receptor 2 and hERR1.
RNA polymerase II/III transcription specificity determined by TATA box orientation.
Authors: Wang Y , Stumph W E
Published: May 2006

The TATA box sequence in eukaryotes is located about 25 bp upstream of many genes transcribed by RNA polymerase II (Pol II) and some genes transcribed by RNA polymerase III (Pol III). The TATA box is recognized in a sequence-specific manner by the TATA box-binding protein (TBP), an essential factor involved in the initiation of transcription by all three eukaryotic RNA polymerases. We have investigated the recognition of the TATA box by the Pol II and Pol III basal transcription machinery and its role in establishing the RNA polymerase specificity of the promoter. Artificial templates were constructed that contained a canonical TATA box as the sole promoter element but differed in the orientation of the 8-bp TATA box sequence. As expected, Pol II initiated transcription in unfractionated nuclear extracts downstream of the "forward" TATA box. In distinct contrast, transcription that initiated downstream of the "reverse" TATA box was carried out specifically by Pol III. Importantly, this effect was observed regardless of the source of the DNA either upstream or downstream of the TATA sequence. These findings suggest that TBP may bind in opposite orientations on Pol II and Pol III promoters and that opposite, yet homologous, surfaces of TBP may be utilized by the Pol II and Pol III basal machinery for the initiation of transcription.
Isolation of cDNA clones encoding RICH: a protein induced during goldfish optic nerve regeneration with homology to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases.
Authors: Ballestero R P , Wilmot G R , Leski M L , Uhler M D , Agranoff B W
Published: May 2006

Using data derived from peptide sequencing of p68/70, a protein doublet induced during optic nerve regeneration in goldfish, we have isolated cDNAs that encode RICH (regeneration-induced CNPase homolog) from a goldfish regenerating retina cDNA library. The predicted RICH protein comprises 411 amino acids, possesses a pI of 4.48, and shows significant homology to the mammalian myelin marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase; EC 3.1.4.37). The mRNA encoding RICH was demonstrated, by both Northern blot analysis and RNase protection assays, to be induced as much as 8-fold in regenerating goldfish retinas at 20 days after nerve crush. Analysis of total RNA samples from various tissues showed a broad distribution of RICH mRNA, with the highest levels observed in gravid ovary. The data obtained strongly suggest that RICH is identical or very similar to p68/70. The molecular cloning of RICH provides the means for a more detailed analysis of its function in nerve regeneration. Additionally, the homology of RICH and CNPase suggests that further investigation may provide additional insight into the role of these proteins in the nervous system.
Mouse Col18a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones.
Authors: Muragaki Y , Timmons S , Griffith C M , Oh S P , Fadel B , Quertermous T , Olsen B R
Published: May 2006

We have isolated overlapping cDNAs encoding the N-terminal non-triple-helical region of mouse alpha 1(XVIII) collagen and shown that three different variants of alpha 1(XVIII) collagen exist. Each of the three variants shows characteristic tissue-specific expression patterns. Immunohistochemical studies show positive staining for alpha 1(XVIII) collagen along the basement membrane zones of vessels in the intestinal villi, the choroid plexus, skin, liver, and kidney. Thus, we conclude that alpha 1(XVIII) collagen may interact (directly or indirectly) with components in basement membrane zones or on the basal surface of endothelial/epithelial cells.
The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guérin.
Authors: Reyrat J M , Berthet F X , Gicquel B
Published: May 2006

The ureABC genes of Mycobacterium tuberculosis were cloned. By using a set of degenerate primers corresponding to a conserved region of the urease enzyme (EC 3.5.1.5), a fragment of the expected size was amplified by PCR and was used to screen a M. tuberculosis cosmid library. Three open reading frames with extensive similarity to the urease genes from other organisms were found. The locus was mapped on the chromosome, using an ordered M. tuberculosis cosmid library. A suicide vector containing a ureC gene disrupted by a kanamycin marker (aph) was used to construct a urease-negative Mycobacterium bovis bacillus Calmette-Guérin mutant by allelic exchange involving replacement of the ureC gene with the aph::ureC construct. To our knowledge, allelic exchange has not been reported previously in the slow-growing mycobacteria. Homologous recombination will be an invaluable genetic tool for deciphering the mechanisms of tuberculosis pathogenesis, a disease that causes 3 x 10(6) deaths a year worldwide.
Transcription termination at intrinsic terminators: the role of the RNA hairpin.
Authors: Wilson K S , von Hippel P H
Published: May 2006

Intrinsic termination of transcription in Escherichia coli involves the formation of an RNA hairpin in the nascent RNA. This hairpin plays a central role in the release of the transcript and polymerase at intrinsic termination sites on the DNA template. We have created variants of the lambda tR2 terminator hairpin and examined the relationship between the structure and stability of this hairpin and the template positions and efficiencies of termination. The results were used to test the simple nucleic acid destabilization model of Yager and von Hippel and showed that this model must be modified to provide a distinct role for the rU-rich sequence in the nascent RNA, since a perfect palindromic sequence that is sufficiently long to form an RNA hairpin that could destabilize the entire putative 12-bp RNA-DNA hybrid does not trigger termination at the expected positions. Rather, our results show that both a stable terminator hairpin and the run of 6-8 rU residues that immediately follows are required for effective intrinsic termination and that termination occurs at specific and invariant template positions relative to these two components. Possible structural or kinetic modifications of the simple model are proposed in the light of these findings and of recent results implicating "inchworming" and possible conformational heterogeneity of transcription complexes in intrinsic termination. Thus, these findings argue that the structure and dimensions of the hairpin are important determinants of the termination-elongation decision and suggest that a complete mechanism is likely to involve specific interactions of the polymerase, the RNA terminator hairpin, and, perhaps, the dT-rich template sequence that codes for the run of rU residues at the 3' end of the nascent transcript.
Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites.
Authors: Pommier Y , Kohlhagen G , Kohn K W , Leteurtre F , Wani M C , Wall M E
Published: May 2006

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface.
A general genetic approach in Escherichia coli for determining the mechanism(s) of action of tumoricidal agents: application to DMP 840, a tumoricidal agent.
Authors: Chatterjee P K , Sternberg N L
Published: May 2006

We describe here a simple and easily manipulatable Escherichia coli-based genetic system that permits us to identify bacterial gene products that modulate the sensitivity of bacteria to tumoricidal agents, such as DMP 840, a bisnaphthalimide drug. To the extent that the action of these agents is conserved, these studies may expand our understanding agents is conserved, these studies may expand our understanding of how the agents work in mammalian cells. The approach briefly is to use a library of E. coli genes that are overexpressed in a high copy number vector to select bacterial clones that are resistant to the cytotoxic effects of drugs. AtolC bacterial mutant is used to maximize permeability of cells to hydrophobic organic molecules. By using DMP 840 to model the system, we have identified two genes, designated mdaA and mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located between the parE and parC genes, and encodes a protein of 22 kDa. mdaA maps to E. coli map coordinate 18, is located adjacent to the glutaredoxin (grx) gene, and encodes a protein of 24 kDa. Specific and regulatable overproduction of both of these proteins correlates with DMP 840 resistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. In contrast, overproduction of the MdaA protein produces resistance only to Adriamycin. Based on its drug-resistance properties and its location between genes that encode the two subunits of the bacterial topoisomerase IV, we suggest that mdaB acts by modulating topoisomerase IV activity. The location of the mdaA gene adjacent to grx suggests it acts by a drug detoxification mechanism.
Fusogenic selectivity of the envelope glycoprotein is a major determinant of human immunodeficiency virus type 1 tropism for CD4+ T-cell lines vs. primary macrophages.
Authors: Broder C C , Berger E A
Published: May 2006

We investigated the relationship between the fusion selectivity of the envelope glycoprotein (env) and the tropism of different human immunodeficiency virus type 1 (HIV-1) isolates for CD4+ human T-cell lines vs. primary macrophages. Recombinant vaccinia viruses were prepared encoding the envs from several well-characterized HIV-1 isolates with distinct cytotropisms. Cells expressing the recombinant envs were mixed with various CD4+ partner cell types; cell fusion was monitored by a quantitative reporter gene assay and by syncytia formation. With CD4+ continuous cell lines as partners (T-cell lines, HeLa cells expressing recombinant CD4), efficient fusion occurred with the envs from T-cell line-tropic isolates (IIIB, LAV, SF2, and RF) but not with the envs from macrophage-tropic isolates (JR-FL, SF162, ADA, and Ba-L). The opposite selectivity pattern was observed with primary macrophages as cell partners; stronger fusion occurred with the envs from the macrophage-tropic than from the T-cell line-tropic isolates. All the envs showed fusion activity with peripheral blood mononuclear cells as partners, consistent with the ability of this cell population to support replication of all the corresponding HIV-1 isolates. These fusion selectivities were maintained irrespective of the cell type used to express env, thereby excluding a role for differential host cell modification. We conclude that the intrinsic fusion selectivity of env plays a major role in the tropism of a HIV-1 isolate for infection of CD4+ T-cell lines vs. primary macrophages, presumably by determining the selectivity of virus entry and cell fusion.
Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain.
Authors: DiSanto J P , Müller W , Guy-Grand D , Fischer A , Rajewsky K
Published: May 2006

The interleukin 2 receptor gamma chain (IL-2R gamma) is a component of the receptors for IL-2, IL-4, IL-7, and IL-15. Mutations in IL-2R gamma in man appear responsible for the X chromosome-linked immunodeficiency SCIDX1, characterized by a defect in T-cell and natural killer (NK)-cell differentiation with the presence of poorly functioning B cells. To explore at which level IL-2R gamma affects lymphoid development in vivo, we have analyzed mice derived from embryonic stem (ES) cells with mutant IL-2R gamma loci generated by Cre/loxP-mediated recombination. In the peripheral blood of chimeric animals, lymphoid cells derived from IL-2R gamma- ES cells were not detected, although control ES cells carrying an IL-2R gamma gene with embedded loxP sites gave rise to T-, B-, and NK-cell lineages. Germline IL-2R gamma-deficient male animals, however, developed some mature splenic B and T cells, although the absolute number of lymphocytes was almost 10-fold reduced. In contrast, there was a complete disappearance of NK cells (over 350-fold reduction). Development of gut-associated intraepithelial lymphocytes was also severely diminished, and Peyer's patches were not detected. In vitro mitogenic responses of thymocytes, IL-4-directed immunoglobulin class switch of splenocytes, and NK activity were defective. Thus, IL-2R gamma facilitates mainstream B- and T-cell generation and function and also appears to be essential for NK-cell development.
Cooperative formation of high-order oligomers by retinoid X receptors: an unexpected mode of DNA recognition.
Authors: Chen H , Privalsky M L
Published: May 2006

Nuclear hormone receptors are ligand-regulated transcription factors that function in metazoan homeostasis and differentiation. We report here that DNA recognition by at least one class of nuclear receptors, the retinoid X receptors (RXRs), can occur through an unanticipated mechanism involving the cooperative formation of protein tetramers and other high-order oligomers. Formation of these oligomeric complexes enables RXRs alpha and gamma, but not beta, to efficiently regulate transcription through response elements poorly recognized by RXR dimers. Thus, oligomer formation plays an important role in determining the specificity of DNA recognition by nuclear receptors and contributes to isoform-dependent differences in gene regulation.
Periodic recurrence of methionines: fossil of gene fusion?
Authors: Kolker E , Trifonov E N
Published: May 2006

As we have recently shown, approximately 20% of proteins are made of uniform size units of approximately 123 aa for eukaryotes and approximately 152 aa for prokaryotes. Such regularity may reflect certain past events in protein evolution by fusion (molecular recombination) of a spectrum of standard-size protein-coding DNA segments--the early genes. Consequently, methionines, as start residues, would mark those locations in proteins that correspond to the DNA recombination sites--the borders between the fused genes. This positional preference of the methionines may still survive as a fossil of the early protein sequence organization. In this study we address the question how methionines are distributed in modern protein sequences. This analysis of eukaryotic sequences shows that methionine residues do preferentially appear at the positions corresponding to the multiples of the unit size, as predicted.
Sphingolipid synthesis as a target for chemotherapy against malaria parasites.
Authors: Lauer S A , Ghori N , Haldar K
Published: May 2006

The human malaria parasite Plasmodium falciparum contains sphingomyelin synthase in its Golgi apparatus and in a network of tubovesicular membranes in the cytoplasm of the infected erythrocyte. Palmitoyl and decanoyl analogues of 1-phenyl-2-acylamino-3-morpholino-1-propanol inhibit the enzyme activity in infected erythrocytes. An average of 35% of the activity is extremely sensitive to these drugs and undergoes a rapid, linear decrease at drug concentrations of 0.05-1 microM. The remaining 65% suffers a slower linear inhibition at drug concentrations ranging from 25 to 500 microM. Evidence is presented that inhibition of the sensitive fraction alone selectively disrupts the appearance of the interconnected tubular network in the host cell cytoplasm, without blocking secretory development at the parasite plasma membrane or in organelles within the parasite, such as the Golgi and the digestive food vacuole. This inhibition also blocks parasite proliferation in culture, indicating that the sensitive sphingomyelin synthase activity as well as the tubovesicular network may provide rational targets for drugs against malaria.
Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.
Authors: Li L , Chin L S , Shupliakov O , Brodin L , Sihra T S , Hvalby O , Jensen V , Zheng D , McNamara J O , Greengard P
Published: May 2006

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.
Gut-specific transcriptional regulatory elements of the carboxypeptidase gene are conserved between black flies and Drosophila.
Authors: Xiong B , Jacobs-Lorena M
Published: May 2006

Millions of people die every year in the tropical world from diseases transmitted by hematophagous insects. Failure of conventional containment measures emphasizes the need for additional approaches, such as transformation of vector insects with genes that restrict vectorial capacity. The availability of an efficient promoter to drive foreign genes in transgenic insects is a necessary tool to test the feasibility of such approach. Here we characterize the putative promoter region of a black fly midgut carboxypeptidase gene and show that these sequences correctly direct the expression of a beta-glucuronidase reporter in Drosophila melanogaster. By histochemical staining and mRNA analysis, we found that the gene is expressed strongly and gut-specifically in the transgenic Drosophila. This gut-specific black fly carboxypeptidase promoter provides a valuable tool for the study of disease vectors.
Hydrophobic cluster analysis predicts an amino-terminal domain of varicella-zoster virus open reading frame 10 required for transcriptional activation.
Authors: Moriuchi H , Moriuchi M , Pichyangkura R , Triezenberg S J , Straus S E , Cohen J I
Published: May 2006

Varicella-zoster virus open reading frame 10 (ORF10) protein, the homolog of the herpes simplex virus protein VP16, can transactivate immediate-early promoters from both viruses. A protein sequence comparison procedure termed hydrophobic cluster analysis was used to identify a motif centered at Phe-28, near the amino terminus of ORF10, that strongly resembles the sequence of the activating domain surrounding Phe-442 of VP16. With a series of GAL4-ORF10 fusion proteins, we mapped the ORF10 transcriptional-activation domain to the amino-terminal region (aa 5-79). Extensive mutagenesis of Phe-28 in GAL4-ORF10 fusion proteins demonstrated the importance of an aromatic or bulky hydrophobic amino acid at this position, as shown previously for Phe-442 of VP16. Transactivation by the native ORF10 protein was abolished when Phe-28 was replaced by Ala. Similar amino-terminal domains were identified in the VP16 homologs of other alphaherpesviruses. Hydrophobic cluster analysis correctly predicted activation domains of ORF10 and VP16 that share critical characteristics of a distinctive subclass of acidic activation domains.
Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.
Authors: Link C D
Published: May 2006

Transgenic Caenorhabditis elegans nematodes have been engineered to express potentially amyloidic human proteins. These animals contain constructs in which the muscle-specific unc-54 promoter/enhancer of C. elegans drives the expression of the appropriate coding regions derived from human cDNA clones. Animals containing constructs expressing the 42-amino acid beta-amyloid peptide (derived from human amyloid precursor protein cDNA) produce muscle-specific deposits immunoreactive with anti-beta-amyloid polyclonal and monoclonal antibodies. A subset of these deposits also bind the amyloid-specific dye thioflavin S, indicating that these deposits have the tinctural characteristics of classic amyloid. Co-expression of beta-peptide and transthyretin, a protein implicated in preventing the formation of insoluble beta-amyloid, leads to a dramatic reduction in the number of dye-reactive deposits. These results suggest that this invertebrate model may be useful for in vivo investigation of factors that modulate amyloid formation.
Mutation of conserved negatively charged residues in the S2 and S3 transmembrane segments of a mammalian K+ channel selectively modulates channel gating.
Authors: Planells-Cases R , Ferrer-Montiel A V , Patten C D , Montal M
Published: May 2006

Voltage-gated channel proteins sense a change in the transmembrane electric field and respond with a conformational change that allows ions to diffuse across the pore-forming structure. Site-specific mutagenesis combined with electrophysiological analysis of expressed mutants in amphibian oocytes has previously established the S4 transmembrane segment as an element of the voltage sensor. Here, we show that mutations of conserved negatively charged residues in S2 and S3 of a brain K+ channel, thought of as countercharges for the positively charged residues in S4, selectively modulate channel gating without modifying the permeation properties. Mutations of Glu235 in S2 that neutralize or reverse charge increase the probability of channel opening and the apparent gating valence. In contrast, replacements of Glu272 by Arg or Thr268 by Asp in S3 decrease the open probability and the apparent gating valence. Residue Glu225 in S2 tolerated replacement only by acidic residues, whereas Asp258 in S3 was intolerant to any attempted change. These results imply that S2 and S3 are unlikely to be involved in channel lining, yet, together with S4, may be additional components of the voltage-sensing structure.
Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.
Authors: Cosentino G P , Venkatesan S , Serluca F C , Green S R , Mathews M B , Sonenberg N
Published: May 2006

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.
Expression of lactoferrin receptors is increased in the mesencephalon of patients with Parkinson disease.
Authors: Faucheux B A , Nillesse N , Damier P , Spik G , Mouatt-Prigent A , Pierce A , Leveugle B , Kubis N , Hauw J J , Agid Y
Published: May 2006

The degeneration of nigral dopaminergic neurons in Parkinson disease is believed to be associated with oxidative stress. Since iron levels are increased in the substantia nigra of parkinsonian patients and this metal catalyzes the formation of free radicals, it may be involved in the mechanisms of nerve cell death. The cause of nigral iron increase is not understood. Iron acquisition by neurons may occur from iron-transferrin complexes with a direct interaction with specific membrane receptors, but recent results have shown a low density of transferrin receptors in the substantia nigra. To investigate whether neuronal death in Parkinson disease may be associated with changes in a pathway supplementary to that of transferrin, lactoferrin (lactotransferrin) receptor expression was studied in the mesencephalon. In this report we present evidence from immunohistochemical staining of postmortem human brain tissue that lactoferrin receptors are localized on neurons (perikarya, dendrites, axons), cerebral microvasculature, and, in some cases, glial cells. In parkinsonian patients, lactoferrin receptor immunoreactivity on neurons and microvessels was increased and more pronounced in those regions of the mesencephalon where the loss of dopaminergic neurons is severe. Moreover, in the substantia nigra, the intensity of immunoreactivity on neurons and microvessels was higher for patients with higher nigral dopaminergic loss. These data suggest that lactoferrin receptors on vulnerable neurons may increase intraneuronal iron levels and contribute to the degeneration of nigral dopaminergic neurons in Parkinson disease.
The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen.
Authors: Papayannopoulou T , Craddock C , Nakamoto B , Priestley G V , Wolf N S
Published: May 2006

Selective lodgement or homing of transplanted hemopoietic stem cells in the recipient's bone marrow (BM) is a critical step in the establishment of long-term hemopoiesis after BM transplantation. However, despite its biologic and clinical significance, little is understood about the process of homing. In the present study, we have concentrated on the initial stages of homing and explored the functional role in vivo of some of the adhesion pathways previously found to mediate in vitro adhesion of hemopoietic cells to cultured BM stroma. We have found that homing of murine hemopoietic progenitors of the BM of lethally irradiated recipients at 3 h after transplant was significantly reduced after pretreatment of the donor cells with an antibody to the integrin very late antigen 4 (VLA4). This inhibition of marrow homing was accompanied by an increase in hemopoietic progenitors circulating in the blood and an increased uptake of these progenitors by the spleen. Similar results were obtained by treatment of the recipients with an antibody to vascular cell adhesion molecule 1 (VCAM-1), a ligand for VLA4. Furthermore, we showed that administration of the same antibodies (anti-VLA4 or anti-VCAM-1) to normal animals causes mobilization of hemopoietic progenitors into blood. These data suggest that hemopoietic cell lodgement in the BM is a regulatable process and can be influenced by VLA4/VCAM-1 adhesion pathway. Although additional molecular pathways are not excluded and may be likely, our data establish VCAM-1 as a BM endothelial addressin, analogous to the role that mucosal addressin cell adhesion molecule (MAdCAM) plays in lymphocyte homing. Whether splenic uptake of hemopoietic progenitors is passive or controlled through different mechanisms remains to be clarified. In addition, we provide experimental evidence that homing and mobilization are related phenomena involving, at least partly, similar molecular pathways.
Calculating the probability of multitaxon evolutionary trees: bootstrappers Gambit.
Authors: Lake J A
Published: May 2006

The reconstruction of multitaxon trees from molecular sequences is confounded by the variety of algorithms and criteria used to evaluate trees, making it difficult to compare the results of different analyses. A global method of multitaxon phylogenetic reconstruction described here, Bootstrappers Gambit, can be used with any four-taxon algorithm, including distance, maximum likelihood, and parsimony methods. It incorporates a Bayesian-Jeffreys'-bootstrap analysis to provide a uniform probability-based criterion for comparing the results from diverse algorithms. To examine the usefulness of the method, the origin of the eukaryotes has been investigated by the analysis of ribosomal small subunit RNA sequences. Three common algorithms (paralinear distances, Jukes-Cantor distances, and Kimura distances) support the eocyte topology, whereas one (maximum parsimony) supports the archaebacterial topology, suggesting that the eocyte prokaryotes are the closest prokaryotic relatives of the eukaryotes.
Simple model of protein folding kinetics.
Authors: Zwanzig R
Published: May 2006

A simple model of the kinetics of protein folding is presented. The reaction coordinate is the "correctness" of a configuration compared with the native state. The model has a gap in the energy spectrum, a large configurational entropy, a free energy barrier between folded and partially folded states, and a good thermodynamic folding transition. Folding kinetics is described by a master equation. The folding time is estimated by means of a local thermodynamic equilibrium assumption and then is calculated both numerically and analytically by solving the master equation. The folding time has a maximum near the folding transition temperature and can have a minimum at a lower temperature.