bluuebunny/crossref_metadata_2025 huggingface dataset:
Deactivation kinetics of the transduction cascade of vision.
Authors: Vuong T M , Chabre M
Published: May 2006

The response of the retinal rod cell to a dim flash lasts less than a second. This phototransduction is mediated by a guanine nucleotide-binding (G) protein cascade in which rhodopsin is the receptor, transducin is the G-protein, and the cGMP-specific phosphodiesterase (PDE) is the effector. Photoexcited rhodopsin activates transducin which in turn activates PDE. For this underlying biochemistry to be kinetically compatible with the photoresponse, both transducin and PDE must be deactivated in subsecond times. We report here direct measurements of their deactivation kinetics. The rate of heat release when transducin and PDE hydrolyze, respectively, GTP and cGMP was measured using time-resolved microcalorimetry. With only GTP present, the heat pulse comes from the activation of transducin and its subsequent deactivation by endogenous GTP hydrolysis. The nonhydrolyzable analog guanine 5'-[gamma-thio]triphosphate was used to distinguish between these two processes: about 40% of the total heat is due to activation. From the time course of the deactivation heat, the active lifetime of transducin is less than 1 s at 22 degrees C. With both GTP and cGMP present, the highly amplified hydrolytic activity of the PDE is responsible for most of the heat produced; its rate of release is directly proportional to the amount of activated PDE. Measurements of this rate at low photoexcitation levels (e.g., 30 molecules of photoexcited rhodopsin per rod) provide much kinetic information about the cascade. Notably, deactivation of the PDE takes 0.6 s (at 23 degrees C) and absolutely requires GTP hydrolysis. This concurs with the subsecond lifetime of active transducin and means that, once GTP hydrolysis has occurred, the hitherto active PDE is quickly inhibited.
Lymphoid organs function as major reservoirs for human immunodeficiency virus.
Authors: Pantaleo G , Graziosi C , Butini L , Pizzo P A , Schnittman S M , Kotler D P , Fauci A S
Published: May 2006

The total number of human immunodeficiency virus type 1 (HIV-1)-infected circulating CD4+ T lymphocytes is considered to be a reflection of the HIV burden at any given time during the course of HIV infection. However, the low frequency of HIV-infected circulating CD4+ T lymphocytes and the low level or absence of plasma viremia in the early stages of infection do not correlate with the progressive immune dysfunction characteristic of HIV infection. In this study, we have determined whether HIV-infected circulating CD4+ T lymphocytes are a correct reflection of the total pool of HIV-infected CD4+ T cells (i.e., HIV burden). To this end, HIV burden has been comparatively analyzed in peripheral blood and lymphoid tissues (lymph nodes, adenoids, and tonsils) from the same patients. The presence of HIV-1 DNA in mononuclear cells isolated simultaneously from peripheral blood and lymphoid tissues of the same patients was determined by polymerase chain reaction amplification. We found that the frequency of HIV-1-infected cells in unfractionated or sorted CD4+ cell populations isolated from lymphoid tissues was significantly higher (0.5-1 log10 unit) than the frequency in peripheral blood. Comparable results were obtained in five HIV seropositive patients in the early stages of disease and in one patient with AIDS. These results demonstrate that a heavy viral load does reside in the lymphoid organs, indicating that they may function as major reservoirs for HIV. In addition, the finding of a heavy viral load in the lymphoid organs of patients in the early stages of disease may explain the progressive depletion of CD4+ T lymphocytes and the immune dysfunction associated with the early stages of HIV infection.
Primary structure and functional expression of a glutaminyl cyclase.
Authors: Pohl T , Zimmer M , Mugele K , Spiess J
Published: May 2006

A glutaminyl cyclase (QC) that is probably involved in the biosynthesis of pyroglutamyl peptides such as gonadotropin-releasing hormone and thyrotropin-releasing hormone has been purified to homogeneity from bovine anterior pituitary. On the basis of N-terminal sequence analysis, a 2088-base-pair cDNA clone was isolated from a bovine anterior pituitary library. From the nucleotide sequence of this clone, the primary structure of a 330-residue protein and a preceding 31-residue prepropeptide sequence was deduced. By transfection of COS-7 monkey cells with a QC cDNA/pCDM8 vector construct, QC activity was expressed. Hybridization with mRNAs of various bovine tissues revealed expression of QC mainly in brain tissue.
Two putative subunits of a peptide pump encoded in the human major histocompatibility complex class II region.
Authors: Bahram S , Arnold D , Bresnahan M , Strominger J L , Spies T
Published: May 2006

The class II region of the human major histocompatibility complex (MHC) may encode several genes controlling the processing of endogenous antigen and the presentation of peptide epitopes by MHC class I molecules to cytotoxic T lymphocytes. A previously described peptide supply factor (PSF1) is a member of the multidrug-resistance family of transporters and may pump cytosolic peptides into the membrane-bound compartment where class I molecules assemble. A second transporter gene, PSF2, was identified 10 kilobases (kb) from PSF1, near the class II DOB gene. The complete sequences of PSF1 and PSF2 were determined from cDNA clones. The translation products are closely related in sequence and predicted secondary structure. Both contain a highly conserved ATP-binding fold and share 25% homology in a hydrophobic domain with a tentative number of eight membrane-spanning segments. Based on the principle dimeric organization of these two domains in other transporters, PSF1 and PSF2 may function as complementary subunits, independently as homodimers, or both. Taken together with previous genetic evidence, the coregulation of PSF1 and PSF2 by gamma interferon and the to-some-degree coordinate transcription of these genes suggest a common role in peptide-loading of class I molecules, although a distinct function of PSF2 cannot be ruled out.
Profound alteration in an alpha beta T-cell antigen receptor repertoire due to polymorphism in the first complementarity-determining region of the beta chain.
Authors: Gahm S J , Fowlkes B J , Jameson S C , Gascoigne N R , Cotterman M M , Kanagawa O , Schwartz R H , Matis L A
Published: May 2006

Amino acid residues that are critical in maintaining the framework structure of immunoglobulin heavy- and light-chain variable (V) regions are strongly conserved in the V alpha and V beta proteins of the alpha beta T-cell antigen receptor (TCR alpha beta). Consequently, it has been proposed that TCR alpha beta has a conformation similar to that of an immunoglobulin Fab fragment and that the regions of the TCR homologous to the three immunoglobulin complementarity-determining regions (CDRs 1, 2, and 3) bind to the peptide antigen-major histocompatibility complex (MHC) molecule ligand. A single amino acid substitution in the predicted CDR1 of the V beta 3 protein of certain mouse strains dramatically altered TCR alpha beta usage in an antigen-specific MHC-restricted immune response but did not abrogate V beta 3 specificity for the superantigens minor lymphocyte stimulatory locus (Mls)c and staphylococcal enterotoxin A (SEA). The results confirm the importance of the V beta CDR1 in antigen-MHC molecule recognition, supporting the Fab-like structural model of TCR alpha beta, and provide further evidence that conventional antigen-MHC recognition and superantigen recognition are mediated by distinct regions of the TCR beta chain. They also suggest that allelic polymorphism may be a significant source of diversity in the TCR repertoire.
Defective membrane expression of human growth hormone (GH) receptor causes Laron-type GH insensitivity syndrome.
Authors: Duquesnoy P , Sobrier M L , Amselem S , Goossens M
Published: May 2006

Mutations in the growth hormone receptor (GHR) gene can cause growth hormone (GH) resistance. Given the sequence homology between the extracellular domain of the GHR and a soluble GH-binding protein (GH-BP), it is remarkable that GH-BP binding activity is absent from the serum of patients with Laron-type GH insensitivity, a hereditary form of severe dwarfism. We have previously identified a mutation within the extracellular domain of this receptor, replacing phenylalanine by serine at position 96 of the mature protein, in a patient with Laron syndrome. We have now investigated the effect of this Phe----Ser substitution on hormone binding activity by expressing the total human GHR cDNA and mutant form in eukaryotic cells. The wild-type protein expressed was able to bind GH but no plasma membrane binding was detectable on cells transfected with the mutant cDNA; this was also the case of cells transfected with a Phe96----Ala mutant cDNA, suggesting that the lack of binding activity is not due to a posttranslational modification of serine. Examination of the variant proteins in subcellular fractions revealed the presence of specific GH binding activity in the lysosomal fraction, whereas immunofluorescence studies located mutant proteins in the cytosol. Our findings suggest that these mutant GHRs fail to follow the correct intracellular transport pathway and underline the potential importance of this phenylalanine residue, which is conserved among the GH, prolactin, and erythropoietin receptors that belong to the same cytokine receptor superfamily.
Mutant profiles of selectable genetic elements.
Authors: Wurst H , Pohl F M
Published: May 2006

A method is presented that allows simultaneous analysis of the effects of all possible point mutations within a specific mutation window of at least 50 base pairs on a DNA fragment that codes for a selectable function. It relies on the detection of mismatched base pairs with hydroxylamine and osmium tetroxide. A mutant plasmid library of randomly distributed point mutations within the lacZ' gene of Escherichia coli was selected for functional alpha-complementation by growth on lactose. The DNA fragments of the selected and unselected library were each heat denatured and again renatured, thereby generating a randomly distributed set of all possible mismatches within the mutagenesis window. Cytidine-containing mismatches were then detected with hydroxylamine, and thymidine-containing mismatches were detected with osmium tetroxide. When this procedure was performed for both DNA strands, all mismatches could be detected. A comparison of the results of the unselected and selected library leads to an estimation of the effects of each detectable mutation on alpha-complementation in vivo. This method, called "mutant profiling," should be applicable to all selectable genetic elements.
Wild-type p53 can down-modulate the activity of various promoters.
Authors: Ginsberg D , Mechta F , Yaniv M , Oren M
Published: May 2006

The wild-type (wt) p53 protein is the product of a tumor suppressor gene that is a frequent target for inactivation in many types of tumors. The nuclear localization of the protein, as well as additional features, suggest that it may be involved in the regulation of gene expression. To explore this possibility, the effects of overproduced wt p53 were investigated in a number of systems. Induction of growth arrest via the antiproliferative effect of wt p53 greatly impaired the ability of cells to exhibit an increase in c-fos mRNA upon serum stimulation. Experiments in which cells were cotransfected with p53 expression plasmids together with a reporter gene linked to various promoters revealed that wt p53 could effectively reduce transcription from a series of promoters derived from serum-inducible genes, but not from a major histocompatibility complex gene. The p53-mediated repression of c-fos gene expression occurred even in the presence of cycloheximide. Kinetic studies indicate that the effect of wt p53 is rapid, rather than representing a secondary consequence of growth arrest. These findings support a role for p53 in transcriptional regulation, perhaps by reducing the expression of genes that are needed for ongoing cell proliferation.
Conversion of the E1A Cys4 zinc finger to a nonfunctional His2,Cys2 zinc finger by a single point mutation.
Authors: Webster L C , Zhang K , Chance B , Ayene I , Culp J S , Huang W J , Wu F Y , Ricciardi R P
Published: May 2006

Trans-activation by the adenovirus E1A 289R protein requires a zinc finger defined by Cys-154, Cys-157, Cys-171, and Cys-174. Whereas individually replacing the four cysteine residues with serines resulted in a loss of transactivation, only three of the Cys----Ser mutants (C157S, C171S, and C174S) lost the ability to bind Zn(II). X-ray absorption fine structure analysis revealed that, in the wild-type protein, Zn(II) is coordinated by four cysteine residues whereas in the C154S mutant, Zn(II) is coordinated by two histidines and two cysteines. The mutant protein probably retains, as ligands, two cysteines on the right side of the zinc finger (Cys-171 and Cys-174) and recruits two of the four histidines on the left side (His-149, His-152, His-158, and His-160), despite the presence of Cys-157. This finding may shed light on the general structural requirements of zinc fingers.
Three-dimensional structure of the Escherichia coli phosphocarrier protein IIIglc.
Authors: Worthylake D , Meadow N D , Roseman S , Liao D I , Herzberg O , Remington S J
Published: May 2006

The crystal structure of a proteolytically modified form of the Escherichia coli phosphocarrier and signal transducing protein IIIglc has been determined by multiple isomorphous and molecular replacement. The model has been refined to an R-factor of 0.166 for data between 6- and 2.1-A resolution with an rms deviation of 0.020 A from ideal bond lengths and 3.2 degrees from ideal bond angles. The molecule is a beta-sheet sandwich, with six antiparallel strands on either side. Several short distorted helices line the periphery of the active site, which is a shallow extremely hydrophobic depression approximately 18 A in diameter near the center of one face. The side chains of the active site histidine residues 75 and 90 face each other at the center of the depression, with the N3 positions exposed to solvent, separated by 3.3 A in an excellent position to form adducts with phosphate. Chloroplatinate forms a divalent adduct with both histidyl side chains, suggesting that the phosphodonor reaction might proceed through a similar transition state. The hydrophobic patch forms the primary crystal contact, suggesting a mode of association of IIIglc with other components of the phosphoenolpyruvate-dependent phosphotransferase system.
Characterization of the interaction of plant transcription factors using a bacterial repressor protein.
Authors: Frohberg C , Heins L , Gatz C
Published: May 2006

Transcription initiation from a eukaryotic polymerase II promoter requires a functional interaction of regulatory transcriptional activators with at least one of the basal transcription factors binding in the vicinity of the TATA box. To characterize this type of interaction in vivo, we have inserted the bacterial Tet repressor-operator complex in nine different positions between an enhancer element (as-1) and the TATA box of the cauliflower mosaic virus (CaMV) 35S RNA promoter. A direct contact between the transcriptional activator ASF-1, which binds to as-1, and the transcriptional machinery should be affected by a repressor protein bound between them, as the spacing of only 34 base pairs (bp) between as-1 and the TATA box is too short to allow looping of the DNA around the repressor. In each construct, the distance of 34 bp was kept constant, while the position of the 19-bp tet operator relative to the TATA box differed by 2 bp. Thus, the position of the Tet repressor relative to the plant transcription factors was consecutively changed by 72 degrees, which allowed us to investigate whether repression depended on the stereospecific alignment of the repressor with the transcription factors. Binding of the Tet repressor to the operator blocked transcription only when the operator was inserted less tha 5 bp from the TATA box. In all other promoter derivatives, no inhibitory effect of the repressor was observed, which suggests that ASF-1 does not directly interact with the general transcription machinery.
Characterization of the two size forms of the alpha 1 subunit of skeletal muscle L-type calcium channels.
Authors: De Jongh K S , Warner C , Colvin A A , Catterall W A
Published: May 2006

The molecular properties of two size forms of the alpha 1 subunit of purified skeletal muscle calcium channels were analyzed. The minor, full-length, form, alpha 1(212), was found to have an apparent molecular mass of 214 kDa by Ferguson plot analysis, while the major, truncated, form, now designated alpha 1(190), had an apparent molecular mass of 193 kDa. Antibody mapping of the C-terminal region of alpha 1(190) with 10 anti-peptide antibodies placed the C terminus between residues 1685 and 1699. Three consensus sites for cAMP-dependent protein phosphorylation are present in the C-terminal region of alpha 1(212) but not in alpha 1(190), and they may be important for the regulation of the ion conductance activity of the calcium channel.
The plastid genome of Cryptomonas phi encodes an hsp70-like protein, a histone-like protein, and an acyl carrier protein.
Authors: Wang S L , Liu X Q
Published: May 2006

The plastid genome of Cryptomonas phi, a cryptomonad alga, contains three genes that have not previously been found in any organellar genome. Each of these genes encodes a functional class of organellar gene product not previously reported. The first gene, dnaK, encodes a polypeptide of the hsp70 heat shock protein family. The predicted amino acid sequence of the DnaK protein is 54% identical to that of the Escherichia coli hsp70 protein (DnaK), 50-53% identical to that of two nucleus-encoded mitochondrial hsp70 proteins, and 43-46% identical to that of several eukaryotic cytoplasmic members of the hsp70 protein family. The second gene, hlpA, encodes a polypeptide resembling bacterial histone-like proteins. The predicted amino acid sequence of the HlpA protein is 25-53% identical to that of several bacterial histone-like proteins, and the identity increases to 39-76% over a conserved region corresponding to the long arm that binds DNA. The third gene, acpA, encodes an acyl carrier protein, which is a key cofactor in the synthesis and metabolism of fatty acids. Its predicted amino acid sequence is 36-59% identical to that of eubacterial and plant chloroplast (nucleus-encoded) acyl carrier proteins.
Protein kinase C activates an H+ (equivalent) conductance in the plasma membrane of human neutrophils.
Authors: Nanda A , Grinstein S
Published: May 2006

The rate of metabolic acid generation by neutrophils increases greatly when they are activated. Intracellular acidification is prevented in part by Na+/H+ exchange, but a sizable component of H+ extrusion persists in the nominal absence of Na+ and HCO3-. In this report we determined the contribution to H+ extrusion of a putative H+ conductive pathway and its mode of activation. In unstimulated cells, H+ conductance was found to be low and unaffected by depolarization. An experimental system was designed to minimize the metabolic acid generation and membrane potential changes associated with neutrophil activation. By using this system, beta-phorbol esters were shown to increase the H+ (equivalent) permeability of the plasma membrane. The direction of the phorbol ester-induced fluxes was dictated by the electrochemical H+ gradient. Moreover, the parallel migration of a counterion through a rheogenic pathway was necessary for the displacement of measurable amounts of H+ equivalents across the membrane. These findings suggest that the H+ flux is conductive. The effect of beta-phorbol esters was mimicked by diacylglycerol and mezerein and was blocked by staurosporine, whereas alpha-phorbol esters were ineffective. Together, these findings indicate that stimulation of protein kinase C induces the activation of an H+ conductance in the plasma membrane of human neutrophils. Preliminary evidence for activation of a separate, bafilomycin A1-sensitive H+ extrusion mechanism, likely a vacuolar type H(+)-ATPase, is also presented.
Crystals of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase with a monoclonal antibody Fab fragment and double-stranded DNA diffract x-rays to 3.5-A resolution.
Authors: Jacobo-Molina A , Clark A D , Williams R L , Nanni R G , Clark P , Ferris A L , Hughes S H , Arnold E
Published: May 2006

Two crystal forms of complexes have been grown that contain human immunodeficiency virus type 1 reverse transcriptase and a monoclonal antibody Fab fragment. One of the crystal forms (form II, space group P3112, a = 168.7 A, c = 220.3 A) diffracts x-rays to 3.5-A resolution and appears suitable for moderate-resolution structure determination. The form II crystals have the unusual property that their maximum resolution of diffraction and resistance to radiation damage are enhanced by either crystallization in the presence of or soaking with double-stranded DNA primer-template mimics. These crystals may permit structural studies of catalytically relevant complexes and eventually enable us to experimentally observe successive steps in the reverse transcription process.
Reconstitution of tracheal grafts with a genetically modified epithelium.
Authors: Engelhardt J F , Allen E D , Wilson J M
Published: May 2006

A rational approach to the development of gene therapies for cystic fibrosis requires a better understanding of the cellular targets for gene transfer in the airway epithelium. We have used recombinant retroviruses to study the dynamics and lineage relationships of a regenerating rat tracheal epithelium. Primary cultures of tracheal epithelial cells were exposed to lacZ-transducing retroviruses and subsequently seeded into denuded trachea that were implanted into BALB/c (nu/nu) mice. The grafts developed a fully differentiated mucociliary epithelium containing large clones of lacZ-expressing cells with virtually all cell types represented within each clone. These data are most consistent with gene transfer into a putative progenitor cell that is capable of extensive self renewal and pleuripotent development. Vector-specific variation in transgene expression was noted in the various cell types.
Human immunodeficiency virus type 1 mutants resistant to nonnucleoside inhibitors of reverse transcriptase arise in tissue culture.
Authors: Richman D , Shih C K , Lowy I , Rose J , Prodanovich P , Goff S , Griffin J
Published: May 2006

We have recently described a nonnucleoside compound that specifically inhibits the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS. This compound, nevirapine (BI-RG-587), interacts with highly conserved tyrosine residues at positions 181 and 188 in the reverse transcriptase to inhibit the recombinant enzyme and virus replication in cell culture with 50% inhibitory concentrations in the 40 nM range. HIV-1 variants resistant to nevirapine emerged with passage in cell culture in the presence of drug. This resistant phenotype was stable with continued passage in the absence of drug. These mutants had a substitution of cysteine for the tyrosine at position 181. Introduction of this mutation into the recombinant enzyme increased the inhibitory concentration of nevirapine 100-fold. Substitution of cysteine for tyrosine at residue 181 into the wild-type viral genome conferred a similar reduction in susceptibility to nevirapine. Mutants were also resistant to a tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione derivative and two 6-phenylthiouracil derivatives but retained their sensitivity to the other reverse transcriptase inhibitors, 3'-azido-3'-deoxythymidine and foscarnet.
Isolation and characterization of a dideoxyguanosine triphosphate-resistant mutant of human immunodeficiency virus reverse transcriptase.
Authors: Prasad V R , Lowy I , de los Santos T , Chiang L , Goff S P
Published: May 2006

The appearance of drug-resistant strains of viral pathogens is a major difficulty confounding current efforts to block viral infections. The identification and analysis of mutations responsible for drug resistance can provide important clues helpful in understanding the mechanisms of resistance and in the eventual development of better therapies. We have used a direct screening method to scan libraries of mutagenized genes encoding the reverse transcriptase of human immunodeficiency virus type 1, and have recovered a variant enzyme that is resistant to the chain-terminator inhibitor 2',3'-dideoxyguanosine triphosphate. The single substitution mutation in this variant conferred broad crossresistance to a variety of other antiviral compounds currently in clinical trials. Virus carrying the mutation was fully infectious in cultured human lymphocytes. The replication of the mutant virus was highly resistant to phosphonoformic acid but did not show increased resistance to the prodrug dideoxyguanosine.
Intracellular replication is essential for the virulence of Salmonella typhimurium.
Authors: Leung K Y , Finlay B B
Published: May 2006

Salmonella typhimurium is a facultative intracellular parasite, capable of penetrating, surviving, and multiplying within diverse eukaryotic cell types, including epithelial and phagocytic cells. We have been studying intracellular replication of S. typhimurium and found that it is essential in the pathogenesis of this bacterium. A total of 45,000 independent mini-Mu MudJ transposon mutants in S. typhimurium SL1344 were screened in Madin-Darby canine kidney (MDCK) epithelial cells with a beta-lactam, cefotaxime, to enrich for mutants defective for intracellular replication. Ten different auxotrophic (purine, pyrimidine, purine/methionine, and valine/isoleucine) and three prototrophic replication-defective mutants (Rep-) were identified. All Rep- mutants showed no differences in aerobic and anaerobic growth patterns, motility, serum sensitivity, mouse macrophage survival, iron uptake, and phosphate requirements. All Rep- mutants were unable to multiply inside MDCK, HeLa, and Caco-2 epithelial cells. When required nutrients for various auxotrophs were supplemented, auxotrophs then replicated inside MDCK cells. Although the parental strain multiplies in large vacuoles inside MDCK cells that distort the host cells, MDCK cells infected with the Rep- mutants appeared relatively normal and few bacteria were seen inside vacuoles. The purine auxotrophs and the three prototrophic Rep- mutants were highly attenuated in mice, and oral and intraperitoneal LD50 levels were 3 to 4 orders of magnitude higher than the wild type level. The three prototrophs were invasive and persisted in the murine organs such as livers and spleens for at least 3 weeks. Therefore, these prototrophic genes are needed for intracellular replication and are essential to the virulence of S. typhimurium.
High-resolution optical imaging of functional brain architecture in the awake monkey.
Authors: Grinvald A , Frostig R D , Siegel R M , Bartfeld E
Published: May 2006

Optical imaging of the functional architecture of cortex, based on intrinsic signals, is a useful tool for the study of the development, organization, and function of the living mammalian brain. This relatively noninvasive technique is based on small activity-dependent changes of the optical properties of cortex. Thus far, functional imaging has been performed only on anesthetized animals. Here we establish that this technique is also suitable for exploring the brain of awake behaving primates. We designed a chronic sealed chamber and mounted it on the skull of a cynomolgus monkey (Macaca fascicularis) over the primary visual cortex to permit imaging through a transparent glass window. Restriction of head position alone was sufficient to eliminate movement noise in awake monkey imaging experiments. High-resolution imaging of the ocular dominance columns and the cytochrome oxidase blobs was achieved simply by taking pictures of the exposed cortex when the awake monkey was viewing video movies alternatively with each eye. Furthermore, the functional maps could be obtained without synchronization of the data acquisition to the animal's respiration and the electrocardiogram. The wavelength dependency and time course of the intrinsic signal were similar in anesthetized and awake monkeys, indicating that the signal sources were the same. We therefore conclude that optical imaging is well suited for exploring functional organization related to higher cognitive brain functions of the primate as well as providing a diagnostic tool for delineating functional cortical borders and assessing proper functions of human patients during neurosurgery.
Structural analysis of the interaction between the human immunodeficiency virus Rev protein and the Rev response element.
Authors: Kjems J , Brown M , Chang D D , Sharp P A
Published: May 2006

The specific interaction between a defined structural element of the human immunodeficiency virus mRNA (RRE, the Rev response element) and the virus-encoded protein Rev has been implicated in the regulation of the export of unspliced or singly spliced mRNA from the nucleus to the cytoplasm. Rev protein was expressed and purified from insect cells using the baculovirus expression system. Chemical and RNase probes were used to analyze the structure of the RRE and the regions involved in Rev binding. Increased reactivity to single-strand-specific probes of nucleotides in two helical domains indicates that Rev binding induces conformational changes in the RRE. Binding of Rev to the RRE primarily protects helical segments and adjacent nucleotides in domain II. A Rev unit binding site is proposed that consists of a six-base-pair helical segment and three adjacent nucleotides. The data also suggest that multiple Rev proteins bind to repeated structural elements of the RRE.
Measuring time to the doctorate: reinterpretation of the evidence.
Authors: Bowen W G , Lord G , Sosa J A
Published: May 2006

There has been increasing concern that the length of time it takes to earn a doctorate in this country has increased dramatically over the last 20 years. The regularly cited evidence--organized by the year in which recipients of doctorates were awarded their degrees--is seriously misleading, however. The application of stable population theory to the problem suggests that the steady fall in the sizes of entering cohorts to graduate school has inflated both the measure of the absolute level of median time to degree and the increase in time to degree. When the same underlying data are reorganized by the year in which recipients of doctorates received their baccalaureate, the statistical bias is eliminated, and the median total time to degree in the humanities is shown to have risen 15-20% rather than the reported 40%.
Photoreceptor peripherin is the normal product of the gene responsible for retinal degeneration in the rds mouse.
Authors: Connell G , Bascom R , Molday L , Reid D , McInnes R R , Molday R S
Published: May 2006

Retinal degeneration slow (rds) is a retinal disorder of an inbred strain of mice in which the outer segment of the photoreceptor cell fails to develop. A candidate gene has recently been described for the rds defect [Travis, G. H., Brennan, M. B., Danielson, P. E., Kozak, C. & Sutcliffe, J. G. (1989) Nature (London) 338, 70-73]. Neither the identity of the normal gene product nor its intracellular localization had been determined. We report here that the amino acid sequence of the bovine photoreceptor-cell protein peripherin, which was previously localized to the rim region of the photoreceptor disk membrane, is 92.5% identical to the sequence of the mouse protein encoded by the normal rds gene. The differences between the two sequences can be attributed to species variation. Monoclonal antibodies were used with Western blot analysis to localize the wild-type mouse peripherin/rds protein to isolated mouse rod outer segments and to show that it, like bovine peripherin, exists as two subunits linked by one or more disulfide bonds. The relative amounts of peripherin/rds protein and rhodopsin in retinal extracts of normal and rds mutant mice were also compared. Identification of peripherin as the protein encoded by the normal rds gene and its localization to membranes of rod outer segments will serve as a basis for studies directed toward defining the role of this protein in the morphogenesis and maintenance of the outer segment and toward understanding the mechanism by which the rds mutation causes retinal degeneration.
Direct and synergistic effects of interleukin 11 on murine hemopoiesis in culture.
Authors: Musashi M , Yang Y C , Paul S R , Clark S C , Sudo T , Ogawa M
Published: May 2006

We have examined the effects of a stromal cell-derived cytokine designated interleukin 11 (IL-11) on the proliferation of murine hemopoietic progenitors in methylcellulose culture. COS cell-conditioned medium containing IL-11 supported formation of granulocyte/macrophage colonies and a small number of multilineage colonies including blast cell colonies in cultures of marrow cells from normal mice. When tested with marrow cells harvested 2 days after injection of 5-fluorouracil at 150 mg/kg, IL-11 enhanced interleukin 3-dependent colony formation, whereas IL-11 alone supported only scant colony formation. Serial observations (mapping studies) of cultures of post-5-fluorouracil spleen cells indicated that the mechanism of the synergistic effect of IL-11 is to shorten the dormant period of stem cells, an effect very similar to that of interleukin 6. When pooled blast cells were plated into medium containing IL-11 and erythropoietin, only macrophage colonies were observed. Thus, IL-11 can directly support the proliferation of committed macrophage progenitors and, and like interleukin 6 and granulocyte colony-stimulating factor, act synergistically with interleukin 3 to shorten the Go period of early progenitors.
Inhibition of growth of experimental prostate cancer with sustained delivery systems (microcapsules and microgranules) of the luteinizing hormone-releasing hormone antagonist SB-75.
Authors: Korkut E , Bokser L , Comaru-Schally A M , Groot K , Schally A V
Published: May 2006

Inhibitory effects of the sustained delivery systems (microcapsules and microgranules) of a potent antagonist of luteinizing hormone-releasing hormone N-Ac-[3-(2-naphthyl)-D-alanine1, 4-chloro-D-phenylalanine2, 3-(3-pyridyl)-D-alanine3, D-citrulline6, D-alanine10]LH-RH (SB-75) on the growth of experimental prostate cancers were investigated. In the first experiment, three doses of a microcapsule preparation releasing 23.8, 47.6, and 71.4 micrograms of antagonist SB-75 per day were compared with microcapsules of agonist [D-Trp6]LH-RH liberating 25 micrograms/day in rats bearing Dunning R3327H transplantable prostate carcinoma. During 8 weeks of treatment, tumor growth was decreased by [D-Trp6]LH-RH and all three doses of SB-75 as compared to untreated controls. The highest dose of SB-75 (71.4 micrograms/day) caused a greater inhibition of prostate cancer growth than [D-Trp6]LH-RH as based on measurement of tumor volume and percentage change in tumor volume. Doses of 23.8 and 47.6 micrograms of SB-75 per day induced a partial and submaximal decrease, respectively, in tumor weight and volume. Tumor doubling time was the longest (50 days) with the high dose of SB-75 vs. 15 days for controls. The body weights were unchanged. The weights of testes, seminal vesicles, and ventral prostate were greatly reduced in all three groups that received SB-75, and testosterone levels were decreased to nondetectable values in the case of the two higher doses of SB-75. LH levels were also diminished. Similar results were obtained in the second experiment, in which the animals were treated for a period of 8 weeks with microgranules of SB-75. Therapy with microgranules of SB-75 significantly decreased tumor growth as measured by the final tumor volume, the percentage change from the initial tumor volume, and the reduction in tumor weight. The results indicate that antagonist SB-75, released from sustained delivery systems, can produce a state of chemical castration and effectively inhibit the growth of experimental prostate cancers. The efficacy of the antagonist SB-75 in inhibiting androgen-dependent Dunning prostatic carcinoma and the absence of side effects suggest its possible usefulness for the treatment of hormone-sensitive tumors.
Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function.
Authors: Nagayama Y , Wadsworth H L , Chazenbalk G D , Russo D , Seto P , Rapoport B
Published: May 2006

To define the sites in the extracellular domain of the human thyrotropin (TSH) receptor that are involved in TSH binding and signal transduction we constructed chimeric thyrotropin-luteinizing hormone/chorionic gonadotropin (TSH-LH/CG) receptors. The extracellular domain of the human TSH receptor was divided into five regions that were replaced, either singly or in various combinations, with homologous regions of the rat LH/CG receptor. The chimeric receptors were stably expressed in Chinese hamster ovary cells. The data obtained suggest that the carboxyl region of the extracellular domain (amino acid residues 261-418) and particularly the middle region (residues 171-260) play a role in signal transduction. The possibility is also raised of an interaction between the amino and carboxyl regions of the extracellular domain in the process of signal transduction. With respect to hormone binding, substitution of the entire extracellular domain of the LH/CG receptor for the corresponding region of the TSH receptor resulted in high-affinity human CG binding with complete loss of TSH binding. Surprisingly, however, there was at least one chimera with a substitution at each of the five domains that still retained high-affinity TSH binding. Substitution of residues 1-170 of the TSH receptor with the corresponding region of the LH/CG receptor was associated with the retention of high-affinity TSH binding but ligand specificity was lost in that TSH and human CG could interact functionally with the receptor. In summary, these studies suggest that the middle region and carboxyl half of the extracellular domain of the TSH receptor are involved in signal transduction and that the TSH-binding region is likely to span the entire extracellular domain, with multiple discontinuous contact sites.
Free radical-derived quinone methide mediates skin tumor promotion by butylated hydroxytoluene hydroperoxide: expanded role for electrophiles in multistage carcinogenesis.
Authors: Guyton K Z , Bhan P , Kuppusamy P , Zweier J L , Trush M A , Kensler T W
Published: May 2006

Free radical derivatives of peroxides, hydroperoxides, and anthrones are thought to mediate tumor promotion by these compounds. Further, the promoting activity of phorbol esters is attributed, in part, to their ability to stimulate the cellular generation of oxygen radicals. A hydroperoxide metabolite of butylated hydroxytoluene, 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone (BHTOOH), has previously been shown to be a tumor promoter in mouse skin. BHTOOH is extensively metabolized by murine keratinocytes to several radical species. The primary radical generated from BHTOOH is a phenoxyl radical that can disproportionate to form butylated hydroxytoluene quinone methide, a reactive electrophile. Since electrophilic species have not been previously postulated to mediate tumor promotion, the present study was undertaken to examine the role of this electrophile in the promoting activity of BHTOOH. The biological activities of two chemical analogs of BHTOOH, 4-trideuteromethyl-BHTOOH and 4-tert-butyl-BHTOOH, were compared with that of the parent compound. 4-Trideuteromethyl-BHTOOH and 4-tert-butyl-BHTOOH have a reduced ability or inability, respectively, to form a quinone methide; however, like the parent compound, they both generate a phenoxyl radical when incubated with keratinocyte cytosol. The potency of BHTOOH, 4-trideuteromethyl-BHTOOH, and 4-tert-butyl-BHTOOH as inducers of ornithine decarboxylase, a marker of tumor promotion, was commensurate with their capacity for generating butylated hydroxytoluene quinone methide. These initial results were confirmed in a two-stage tumor promotion protocol in female SENCAR mice. Together, these data indicate that a quinone methide is mediating tumor promotion by BHTOOH, providing direct evidence that an electrophilic intermediate can elicit this stage of carcinogenesis.
Promotion of central cholinergic and dopaminergic neuron differentiation by brain-derived neurotrophic factor but not neurotrophin 3.
Authors: Knüsel B , Winslow J W , Rosenthal A , Burton L E , Seid D P , Nikolics K , Hefti F
Published: May 2006

Recombinant human brain-derived neurotrophic factor (rhBDNF) and neurotrophin 3 (rhNT-3), two recently cloned molecules closely related to nerve growth factor (NGF), were produced from human cDNA expressed in human embryonic kidney cells. The recombinant proteins were tested in cultures of dissociated fetal rat brain cells containing basal forebrain cholinergic neurons. rhBDNF stimulated the differentiation of the cholinergic neurons, similar to NGF, which is well established as a neurotrophic factor for these cells. However, rhBDNF was particularly effective during the first few days in vitro, whereas the stimulatory action of rhNGF was more pronounced later in the development of the cultures. This finding indicates the existence of different time periods of responsiveness of the cholinergic neurons to BDNF and NGF. To assess the selectivity of the effect of rhBDNF on cholinergic neurons, its actions were tested in cultures of ventral mesencephalon containing dopaminergic cells. In contrast to NGF, which does not affect central dopaminergic neurons, rhBDNF increased dopamine uptake activity. The findings suggest that BDNF stimulates survival or differentiation of other cells besides the cholinergic neurons.
A 45-kDa protein antigenically related to band 3 is selectively expressed in kidney mitochondria.
Authors: Ostedgaard L S , Jennings M L , Karniski L P , Schuster V L
Published: May 2006

Anion exchange similar to that catalyzed by erythrocyte band 3 occurs across many nonerythroid cell membranes. To identify anion-exchange proteins structurally related to band 3, we immunoblotted rabbit kidney medullary membrane fractions with anti-band 3 antibodies. Immunoblots using antibodies to the cytoplasmic domain of band 3 revealed cross-reactive proteins in the plasma membrane fraction only. In contrast, two monoclonal antibodies against band 3 membrane domain labeled a 45-kDa protein; further immunoblotting and immunogold studies of membrane fractions and kidney sections using one of the anti-membrane domain antibodies showed that labeling was strongest in mitochondria of H(+)-secreting collecting duct cells. Tissue-to-tissue expression of the 45-kDa mitochondrial protein was variable: kidney medulla greater than heart greater than kidney cortex much greater than liver. We conclude that a 45-kDa protein with immunological cross-reactivity to the erythrocyte band 3 membrane domain is expressed in mitochondria in a highly cell-specific fashion and speculate that the protein may play a role in mitochondrial anion transport.
Inducible proteins binding to the murine thymidine kinase promoter in late G1/S phase.
Authors: Dou Q P , Fridovich-Keil J L , Pardee A B
Published: May 2006

By performing DNase I footprint and band-shift analyses of a 170-base-pair region of the murine thymidine kinase promoter, we identified an inducible DNA binding activity that we named Yi. Yi binding activity was not detected in G0 and G1 extracts, but it was observed as cells crossed the G1/S boundary. Yi proteins bind specifically to a consensus sequence (CCCNCNNNCT) found at three distinct sites in this promoter region. We also observed a murine Sp1 binding activity that was constitutive throughout the cell cycle. We propose that the G1/S-specific Yi binding is important for murine thymidine kinase gene regulation and perhaps also for initiation of DNA synthesis.
In vivo mapping of a sequence required for interference with the yeast killer virus.
Authors: Huan B F , Shen Y Q , Bruenn J A
Published: May 2006

The Saccharomyces cerevisiae viruses are noninfectious double-stranded RNA viruses whose segments are separately encapsidated. A large viral double-stranded RNA (L1; 4580 base pairs) encodes all required viral functions. M1, a double-stranded RNA of 1.9 kilobases, encodes an extracellular toxin (killer toxin) and cellular immunity to that toxin. Some strains contain smaller, S, double-stranded RNAs, derived from M1 by internal deletion. Particles containing these defective interfering RNAs can displace M1 particles by faster replication and thus convert the host strain to a nonkiller phenotype. In this work, we report the development of an assay in which the expression of S plus-strand from an inducible plasmid causes the loss of M1 particles. This assay provides a convenient method for identifying in vivo cis-acting sequences important in viral replication and packaging. We have mapped the sequence involved in interference to a region of 132 base pairs that includes two sequences similar to the viral binding site sequence previously identified in L1 by in vitro experiments.
An antiviral target on reverse transcriptase of human immunodeficiency virus type 1 revealed by tetrahydroimidazo-[4,5,1-jk] [1,4]benzodiazepin-2 (1H)-one and -thione derivatives.
Authors: Debyser Z , Pauwels R , Andries K , Desmyter J , Kukla M , Janssen P A , De Clercq E
Published: May 2006

Screening of pharmacologically acceptable prototype compounds has recently led to the discovery of a series of ultraselective inhibitors of human immunodeficiency virus (HIV)-1 replication, the tetrahydroimidazo[4,5,1-jk] [1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) derivatives. The TIBO compounds completely suppress the formation of proviral DNA in acutely infected cells, as revealed by polymerase chain reaction (PCR) analysis. TIBO derivatives are inhibitory to the reverse transcriptase (RT) of HIV-1 but not that of HIV-2 or other retroviruses. The inhibition is most effective with poly(C)-oligo(dG) as the template/primer, and it is selectively directed against the RNA-dependent DNA polymerase activity and not the accompanying DNA-dependent DNA polymerase and ribonuclease H activity of HIV-1 RT. Kinetic studies point to an uncompetitive inhibition with regard to the template/primer. TIBO compounds are active against HIV-1 replication through a unique interaction with HIV-1 RT. The experimental data indicate the existence of a target on HIV-1 RT that is responsible for the inhibition of replication and a mode of action unrelated to that of previously studied RT inhibitors.