bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies with glycolysis-deficient cells suggest that production of lactic acid is not the only cause of tumor acidity.
Authors: Newell K , Franchi A , Pouysségur J , Tannock I
Published: May 2006

Solid tumors have been observed to develop an acidic extracellular environment, which is believed to occur as a result of lactic acid accumulation produced during aerobic and anaerobic glycolysis. Experiments using glycolysis-deficient ras-transfected Chinese hamster lung fibroblasts have been performed to test the hypothesis that lactic acid production within solid tumors is responsible for the development of tumor acidity. The variant cells have defects in glucose transport and in the glycolytic enzyme phosphoglucose isomerase with 1% activity compared to parental cells. Consequently, the in vitro rate of lactic acid production by variant cells was < 4% compared to parental cells. An in vitro correlation between lactic acid production and acidification of exposure medium was observed for parental and variant cells. Implantation of both cell lines into nude mice led to tumors with minimal difference in growth rate. As expected, variant cells died when exposed to hypoxic conditions in culture, and parental tumors were observed to have a larger fraction of cells resistant to radiation due to hypoxia (27%) than variant tumors (2%). Using pH microelectrodes, parental (n = 12) and variant (n = 12) tumors were observed to have extracellular pH (pHe) values of 6.65 +/- 0.07 and 6.78 +/- 0.04 (mean +/- SE, P = 0.13), respectively, whereas normal muscle had a pHe of 7.29 +/- 0.06 (P < 0.0001 for both cell lines). The lactic acid content of variant tumors was found to be similar to that in serum, whereas parental tumors had lactic acid content that was higher than in serum (P < 0.0001). We conclude that there was no correlation between lactic acid content and acidosis for these tumors derived from ras-transfected fibroblasts. These results provide evidence that the production of lactic acid via glycolysis is not the only mechanism responsible for the development of an acidic environment within solid tumors.
Transcription of the sex-determining region genes Sry and Zfy in the mouse preimplantation embryo.
Authors: Zwingman T , Erickson R P , Boyer T , Ao A
Published: May 2006

We have confirmed the faster growth of male preimplantation mouse embryos. We have also studied the transcription of Y chromosomal genes postulated to have a role in sex determination, using the highly sensitive technique of reverse-transcription polymerase chain reaction at these early stages. We find that two sex-determining region genes, Sry and Zfy, are transcribed during mouse preimplantation development, while the Zfy homologs Zfx and Zfa and a sex-determining region gene originally called A1s9 (now called Ube1y-1) are not. We also show that the anti-Müllerian hormone gene, which contains a Sry consensus binding element in its 5' promoter region, is not transcribed at this time. Developmental curves show that Sry and Zfy are expressed commencing at the two-cell stage. These results suggest that mammalian sex determination starts prior to gonad differentiation.
Casimir light: a glimpse.
Authors: Schwinger J
Published: May 2006

Light emission produced by the reversible collapse of a cavity in a dielectric medium is given an initial, simplified treatment. The agreement between planar and spherical shapes indicates the volume nature of the effect.
Two light-transducing membrane proteins: bacteriorhodopsin and the mammalian rhodopsin.
Authors: Khorana H G
Published: May 2006

Site-directed mutagenesis has provided insight into the mechanisms of action of bacteriorhodopsin and rhodopsin. These studies are summarized here.
A small single-"finger" peptide from the erythroid transcription factor GATA-1 binds specifically to DNA as a zinc or iron complex.
Authors: Omichinski J G , Trainor C , Evans T , Gronenborn A M , Clore G M , Felsenfeld G
Published: May 2006

Sequence-specific DNA binding has been demonstrated for a synthetic peptide comprising only one of the two "finger"-like domains of the erythroid transcription factor GATA-1 (also termed Eryf-1, NF-E1, or GF-1). Quantitative analysis of gel-retardation assays yields a specific association constant of 1.2 x 10(8) M, compared with values of about 10(9) M for the full-length natural GATA-1 protein. By the use of peptides of various lengths, it was possible to delineate the smallest region necessary for specific binding. A single C-terminal finger of the double-finger motif is necessary but not sufficient for sequence-specific interaction. Basic amino acids located C-terminal to the finger (some more than 20 amino acids away) are also essential for tight binding. In addition to demonstrating that zinc is important for the formation of an active binding complex, we show that other ions, notably Fe2+, can fulfill this role. Our results make it clear that the GATA-1 metal binding motif is quite distinct from that found in the steroid hormone family and that GATA-1 is a member of a separate class of DNA binding proteins.
Expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy.
Authors: Yamamoto T , Nakamura T , Noble N A , Ruoslahti E , Border W A
Published: May 2006

Diabetes is now the most common cause of progressive kidney failure leading to dialysis or transplantation. The central pathological feature of diabetic nephropathy is accumulation of extracellular matrix within the glomeruli. The factors in the diabetic milieu responsible for extracellular matrix accumulation are not understood. Here we report that in glomeruli of rats made diabetic there is a slow, progressive increase in the expression of transforming growth factor beta (TGF-beta) mRNA and TGF-beta protein. A key action of TGF-beta is induction of extracellular matrix production, and specific matrix proteins known to be induced by TGF-beta were increased in diabetic rat glomeruli. These proteins include an alternatively spliced form of fibronectin, tenascin, and the proteoglycan biglycan. TGF-beta has not previously been implicated in the matrix accumulation that occurs in the diabetic kidney. Glomeruli from humans with diabetic nephropathy also showed a striking increase in immunoreactive TGF-beta protein and deposition of the special form of fibronectin, whereas glomeruli from normal subjects or from individuals with other glomerular diseases (where extracellular matrix accumulation is not a feature) were negative or barely positive. These results implicate TGF-beta in the pathogenesis of diabetic nephropathy.
Oxygen evolution in photosynthesis: from unicycle to bicycle.
Authors: Shinkarev V P , Wraight C A
Published: May 2006

Flash-induced oxygen evolution in the thylakoids of plants and algae exhibits damped oscillations with period four. These are well described by the S-state model of Kok et al. [Kok, B., Forbush, B. & McGloin, M. (1970) Photochem. Photobiol. 11, 457-475], with damping provided by empirical misses and double hits in the reaction center of photosystem II. Here we apply a mechanistic interpretation of misses as mainly determined by reaction centers that are inactive at the time of the flash due to the presence of either P+ or QA, according to the electron transfer equilibria on the donor and acceptor sides of the reaction center. Calculation of misses on this basis, using known or estimated values of the equilibrium constants for electron transfer between the S states and tyrosine Yz, between Yz and P680, as well as between the acceptor plastoquinones, allows a natural description of the flash number dependence of oxygen evolution. The calculated misses are different for each flash-induced reaction center transition. Identification of this mechanism underlying the miss factor for each transition leads to the recognition of two different reaction sequence cycles of photosystem II, with different transition probabilities, producing an intrinsic heterogeneity of photosystem II activity.
A major histocompatibility complex-linked locus in the rat critically influences resistance to diethylnitrosamine carcinogenesis.
Authors: Melhem M F , Kunz H W , Gill T J
Published: May 2006

Major histocompatibility complex (MHC)-linked deletions in the rat are associated with defects in growth and development and increased susceptibility to chemical carcinogens. The present study maps a locus critical for determining susceptibility to diethylnitrosamine (DEN) carcinogenesis by using two groups of MHC-recombinant rats congenic for the MHC and its linked region. Resistance to DEN segregates with a locus (rcc+) that maps between RT1.E and ft, and its homozygous loss markedly increases susceptibility to DEN. Non-MHC genes do not significantly influence the susceptibility of these strains to DEN. The existence of the rcc locus adds support to our hypothesis that some genes in the MHC-linked region play a major role in both normal and abnormal growth.
A mammalian DNA-binding protein that contains a chromodomain and an SNF2/SWI2-like helicase domain.
Authors: Delmas V , Stokes D G , Perry R P
Published: May 2006

Two overlapping cDNAs that encode a 197-kDa sequence-selective DNA-binding protein were isolated from libraries derived from mouse lymphoid cell mRNA. In addition to a DNA-binding domain, the protein contains both a chromodomain, which occurs in proteins that are implicated in chromatin compaction, and an SNF2/SWI2-like helicase domain, which occurs in proteins that are believed to activate transcription by counteracting the repressive effects of chromatin structure. A Southern blot analysis indicated that this protein, which we have named CHD-1, for chromodomain-helicase-DNA-binding protein, is present in most, if not all, mammalian species. A Northern blot analysis revealed multiple CHD mRNA components that differed both qualitatively and quantitatively among various cell types. The various mRNAs, which are probably produced by alternative RNA processing, could conceivably encode tissue-specific and developmental stage-specific isoforms of the protein. Based on its interesting combination of features, we suspect that CHD-1 plays an important role in gene regulation.
Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis.
Authors: Zheng L , White R H , Cash V L , Jack R F , Dean D R
Published: May 2006

Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme composed of two separately purifiable component proteins encoded by the structural genes nifH, nifD, and nifK. Deletion of the Azotobacter vinelandii nifS gene lowers the activities of both nitrogenase component proteins. Because both nitrogenase component proteins have metallocluster prosthetic groups that are composed of iron- and sulfur-containing cores, this result indicated that the nifS gene product could be involved in the mobilization of the iron or sulfur required for metallocluster formation. In the present work, it is shown that NIFS is a pyridoxal phosphate-containing homodimer that catalyzes the formation of L-alanine and elemental sulfur by using L-cysteine as substrate. NIFS activity is extremely sensitive to thiol-specific alkylating reagents, which indicates the participation of a cysteinyl thiolate at the active site. Based on these results we propose that an enzyme-bound cysteinyl persulfide that requires the release of the sulfur from the substrate L-cysteine for its formation ultimately provides the inorganic sulfide required for nitrogenase metallocluster formation. The recent discovery of nifS-like genes in non-nitrogen-fixing organisms also raises the possibility that the reaction catalyzed by NIFS represents a universal mechanism that involves pyridoxal phosphate chemistry, in the mobilization of the sulfur required for metallocluster formation.
Lethal thalassemia after insertional disruption of the mouse major adult beta-globin gene.
Authors: Shehee W R , Oliver P , Smithies O
Published: May 2006

Thalassemias are hereditary anemias caused by mutations that disturb the normal 1:1 balance of alpha- and beta-globin chains that form hemoglobin. We have disrupted the major adult beta-globin gene (b1) in mouse embryonic stem cells by using homologous recombination to insert selectable sequences into the gene. Mice homozygous for this insertional disruption of the b1 gene (Hbbth-2/Hbbth-2) are severely anemic and die perinatally. In contrast, approximately 60% of mice homozygous for deletion of the same gene (Hbbth-1/Hbbth-1) survive to adulthood and are much less anemic [Skow, L. C., Burkhart, B. A., Johnson, F. M., Popp, R. A., Goldberg, S. Z., Anderson, W. F., Barnett, L. B. & Lewis, S. E. (1983) Cell 34, 1043-1052]. These different phenotypes have implications for the control of beta-globin gene expression.
Data base analysis of protein expression patterns during T-cell ontogeny and activation.
Authors: Hanash S M , Strahler J R , Chan Y , Kuick R , Teichroew D , Neel J V , Hailat N , Keim D R , Gratiot-Deans J , Ungar D
Published: May 2006

We have developed a data base of lymphoid proteins detectable by two-dimensional polyacrylamide gel electrophoresis. The data base contains two-dimensional patterns and derived information pertaining to polypeptide constituents of unstimulated and stimulated mature T cells and immature thymocytes, single-cell-derived T- and B-cell clones, leukemia cells, and lymphoid cell lines. Using this data base, we have compared the protein constituents of mature T cells and immature thymocytes before and after mitotic stimulation. A subset of polypeptides that are induced in mature T cells following mitotic stimulation were found to be constitutively expressed in immature thymocytes. Other polypeptides exhibited differences in their expression between mature and immature thymocytes in a manner unrelated to proliferation. The identity of several constitutively expressed or mitotically induced proteins in lymphoid cells was established by microsequencing. These initial findings point to significant differences in the molecular pathways leading to proliferation between mature and immature T cells. The construction of this database should facilitate further studies of lymphoid differentiation and function.
The nucleotide in position 32 of the tRNA anticodon loop determines ability of anticodon UCC to discriminate among glycine codons.
Authors: Lustig F , Borén T , Claesson C , Simonsson C , Barciszewska M , Lagerkvist U
Published: May 2006

We have investigated the influence of structures in the tRNA anticodon loop and stem on the ability of the anticodon to discriminate among codons. We had previously shown that anticodon UCC, when placed in the structural context of tRNA(Gly1) from Escherichia coli, discriminated efficiently between the glycine codons, as required by the wobble rules. Thus, this anticodon read GGA and GGG but did not read GGU and GGC, whereas in mycoplasma tRNA(Gly), the same anticodon did not discriminate among the glycine codons. We have now determined the reading properties of three constructions based on tRNA(Gly1) containing the anticodon UCC in different structural contexts. In one of these constructs, tRNA(Gly1-ASL), the anticodon loop and stem are the same as in mycoplasma tRNA(Gly). The second construct, tRNA(Gly1-AS), has an anticodon stem identical with the mycoplasma tRNA(Gly), whereas in the last construct, tRNA(Gly1-C32), the only difference from tRNA(Gly1)(UCC) is that the uridine in position 32 of the anticodon loop has been replaced by cytidine. These constructs were tested for ability to read glycine codons in an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each codon. We found that the anticodon UCC, when present in tRNA(Gly1-AS), discriminated among the glycine codons, whereas in the constructs tRNA(Gly1-ASL) and tRNA(Gly1-C32), the same anticodon had lost its ability to discriminate--i.e., it behaved as in mycoplasma tRNA(Gly). These results strongly suggest that nt 32 of the anticodon loop of tRNA(Gly1)(UCC) decisively influences the reading properties of the anticodon UCC.
The role of "indirect" recognition in initiating rejection of skin grafts from major histocompatibility complex class II-deficient mice.
Authors: Auchincloss H , Lee R , Shea S , Markowitz J S , Grusby M J , Glimcher L H
Published: May 2006

In vitro studies have revealed several pathways by which T cells can respond to alloantigens, including CD4+ direct responses to allogeneic class II antigens, CD8+ direct responses to allogeneic class I antigens, and CD4+ "indirect" responses to peptides of alloantigens presented in association with responder class II molecules. In vivo studies of skin graft rejection, however, have so far provided clear evidence for the contribution of only the two direct pathways and not for indirect recognition. We have used major histocompatibility complex class II-deficient mice as donors to test the role of indirect recognition in rejection of skin grafts. Class II-deficient skin was always rejected without delay by normal recipients. Removal of recipient CD8+ cells (to leave the animals dependent on CD4+ function) or depletion of recipient CD4+ cells revealed that CD4+ cells were usually involved and sometimes absolutely required in this rapid rejection. Since the donor grafts lacked class II antigens, the CD4+ cells must have recognized donor antigens presented in association with recipient class II molecules. These results therefore indicate that indirect recognition can initiate rapid skin graft rejection.
Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA.
Authors: Sundquist W I , Heaphy S
Published: May 2006

Retroviruses package two homologous single-stranded RNA genomes within a gag protein-RNA complex. In mature virion particles, the two RNA strands are thought to associate primarily through direct RNA-RNA interactions, although the structural basis for this stable association is unknown. We now report that a 127-nucleotide (nt) HIV-1NL4-3 RNA fragment (positions 732-858) encompassing the 5' end of the gag gene dimerizes spontaneously under high ionic strength conditions in the absence of any protein cofactor. The HIV-1 RNA dimer is dramatically and specifically stabilized by the monovalent cation potassium. Thermal dissociation of the dimer occurs at 80 degrees C in 100 mM K+ (5 mM Mg2+) but at significantly lower temperatures in the presence of either smaller or larger monovalent cations (100 mM Li+, 40 degrees C; 100 mM Na+, 55 degrees C; 100 mM Cs+, 30 degrees C). Deletion analyses of the 3' end of the 127-nt fragment reveal that an HIV-1 RNA fragment as short as 94 nt (732-825) can dimerize spontaneously, but a further 9-base deletion of the purine-rich sequence, GGGGGAGAA from positions 817 through 825, eliminates dimerization. These experimental results support a model in which HIV-1 RNA dimerizes by forming an interstrand quadruple helix stabilized by guanine (and/or purine)-base tetrads in analogy to the well-known dimerization of telomeric DNA. We speculate that this structure may also mediate the association of genomic HIV-1 RNA in vivo, revealing how RNA itself can achieve the self-recognition required for subsequent genetic recombination.
Cognition-activated low-frequency modulation of light absorption in human brain.
Authors: Chance B , Zhuang Z , UnAh C , Alter C , Lipton L
Published: May 2006

Animal model studies indicate light-absorption changes of the exposed animal brain in response to visual stimulation. Here we report observations of red-light absorbance changes, attributable to repetitive blood concentration changes in response to stimulation in the human brain frontal region by a cognitive process. These responses are observed as low-frequency recurrence of changes by Fourier transform analysis and are attributed to blood concentration change stimulated by the increased metabolic rate of brain tissue in cognitive function. A simple, portable dual wavelength spectrophotometer was attached noninvasively to the human forehead to measure the low frequency and power spectra of fluctuations of absorbances attributed to variations of brain blood concentration in the frontal region. The responses are associated with brain activity in responses to problem solving of analogies presented visually that require an associative function in the frontal region. The method of subtraction of test -rest Fourier transforms minimizes the arterial pulse frequency contributions and identifies specific frequencies--for example, 0.8, 1.6, 1.8 Hz in 24 of 28 tests of nine individuals (85%). Tests in which no increased brain activity was elicited (rest-rest) showed small differences. It is concluded that low-frequency recurrences of brain activity linked to blood concentration increases can be detected in human subjects with an optical device of potentially for simplified tests of cognitive function in the 0- to 3-Hz region and with modifications for wider band recordings in localized tissue volumes by time-resolved spectroscopy.
Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene.
Authors: Suber M L , Pittler S J , Qin N , Wright G C , Holcombe V , Lee R H , Craft C M , Lolley R N , Baehr W , Hurwitz R L
Published: May 2006

Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.
Transmission-blocking activity of a chitinase inhibitor and activation of malarial parasite chitinase by mosquito protease.
Authors: Shahabuddin M , Toyoshima T , Aikawa M , Kaslow D C
Published: May 2006

During development in the mosquito midgut, malarial parasites must traverse a chitin-containing peritrophic matrix (PM) that forms around the food bolus. Previously Huber et al. [Huber, M., Cabib, E. & Miller, L. H. (1991) Proc. Natl. Acad. Sci. USA 88, 2807-2810] reported that the parasite secretes a protein with chitinase activity, and they suggested that parasite chitinase (EC 3.2.1.14) plays an important role in the parasite's egress from the blood meal. We found that allosamidin, a specific inhibitor of chitinase, completely blocked oocyst development in vivo and thus blocked malaria parasite transmission. Addition of exogenous chitinase to the blood meal prevented the PM from forming and reversed the transmission-blocking activity of allosamidin. Using exogenous chitinase, we also found that the PM does not limit the number of parasites that develop into oocysts, suggesting that the parasite produces sufficient quantities of chitinase to penetrate this potential barrier. In addition, we found that treatment of parasite chitinase with a diisopropyl fluorophosphate-sensitive trypsinlike protease from the mosquito midgut or endoproteinase Lys-C increased its enzymatic activity. These results suggest that malaria parasite has evolved an intricate mechanism to adapt to the PM and the protease-rich environment of the mosquito midgut.
Efficient transactivation by retinoic acid receptors in yeast requires retinoid X receptors.
Authors: Heery D M , Zacharewski T , Pierrat B , Gronemeyer H , Chambon P , Losson R
Published: May 2006

All-trans and 9-cis retinoic acids are natural derivatives of vitamin A that modulate gene expression as a consequence of binding to nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RXRs form heterodimers with RARs in vitro, and such complexes display enhanced binding affinities for cognate DNA response elements. As yeast is devoid of endogenous RARs and RXRs, we used this organism to investigate whether transactivation in vivo requires RAR/RXR heterodimers. Using a domain-swapping approach, we demonstrate that chimeric RAR alpha 1 and RXR alpha containing the DNA-binding domain of the estrogen receptor activate transcription of a cognate reporter gene in yeast, independently of each other. These activities result from an inducible transcription activation function located in the ligand-binding domains of RAR alpha 1 and RXR alpha and a constitutive activation function located in the A/B region of RAR alpha 1. The inducible activation function of RXR alpha is induced exclusively by 9-cis-retinoic acid in this system. Transactivation of a reporter gene containing a retinoic acid response element by RAR alpha was considerably increased by RXR alpha, even in the absence of ligand. Optimal induction was achieved with 9-cis-retinoic acid, which stimulates the activity of both receptors. This study illustrates the utility of yeast to investigate signal transduction by retinoids in the absence of endogenous RARs, RXRs, and detectable retinoic acid isomerization.
General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia.
Authors: Wang G L , Semenza G L
Published: May 2006

Transcription of the human erythropoietin (EPO) gene is activated in Hep3B cells exposed to hypoxia. Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor whose DNA binding activity is induced by hypoxia in Hep3B cells, and HIF-1 binds at a site in the EPO gene enhancer that is required for hypoxic activation of transcription. In this paper, we demonstrate that HIF-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed. The composition of the HIF-1 DNA binding complex and its isolated DNA binding subunit and the mechanism of HIF-1 activation appear to be similar or identical in EPO-producing and non-EPO-producing cells. Transcription of reporter genes containing the EPO gene enhancer is induced by hypoxia in non-EPO-producing cells and mutations that eliminate HIF-1 binding eliminate inducibility. These results provide evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia.
Cysteine-rich LIM domains of LIM-homeodomain and LIM-only proteins contain zinc but not iron.
Authors: Archer V E , Breton J , Sanchez-Garcia I , Osada H , Forster A , Thomson A J , Rabbitts T H
Published: May 2006

The structure of LIM domains has major implications for transcription because proteins such as Is1-1 contain two LIM domains associated with a homeodomain, and RBTN1/Ttg-1 and RBTN2/Ttg-2 contain two LIM domains but no homeodomain. Conserved cysteine and histidine residues in the LIM domains suggest a metal-binding role. RBTN and Is1-1 LIM proteins have been made in Escherichia coli and insect cell expression systems and their metal content has been determined using atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy. LIM proteins expressed in soluble form contain zinc atoms, whereas bacterial inclusion bodies invariably also have Fe-S clusters. The latter are identified as linear [Fe3S4]+ clusters and appear to result from incorrect metal coordination by E. coli. These studies show that RBTN1, RBTN2, and Is1-1 are metalloproteins that contain zinc but not iron and, therefore, that the LIM domain represents a zinc-binding domain.
The mitogen-regulated protein/proliferin transcript is degraded in primary mouse embryo fibroblast but not 3T3 nuclei: altered RNA processing correlates with immortalization.
Authors: Malyankar U M , Rittling S R , Connor A , Denhardt D T
Published: May 2006

An understanding of what changes occur in the control of gene expression when mammalian cells "spontaneously" immortalize is important to our knowledge of how cancer develops. We describe here an alteration in regulation that occurs when primary mouse embryo fibroblasts (MEFs) are immortalized according to a 3T3 regimen. Mitogen-regulated protein/proliferin mRNA is undetectable in northern blots of RNA from (mortal) MEFs, whereas it is readily detected in immortal 3T3 cell lines derived from the MEFs. Incompletely processed nuclear transcripts of the mitogen-regulated protein/proliferin gene can be detected in MEF RNA preparations by northern blotting and reverse transcriptase polymerase chain reaction analyses, although at roughly half the abundance observed in 3T3 cells. We hypothesize that some attribute of the primary unprocessed transcript determines its assignment to this unique degradative pathway. These results reveal that during passage of MEFs according to a 3T3 regimen the ability of the primary cells to suppress the expression of certain genes by degrading the nuclear transcript is lost concomitantly with immortalization.
A conserved stem-loop structure in the 3' untranslated region of procyclin mRNAs regulates expression in Trypanosoma brucei.
Authors: Hehl A , Vassella E , Braun R , Roditi I
Published: May 2006

African trypanosomes that cycle between mammalian hosts and the tsetse fly vector must be poised to survive in different environments. The control of stage-specific gene expression is undoubtedly one of the keys to successful adaptation, but no regulatory elements have been defined to date. Procyclins (also known as procyclic acidic repetitive proteins) are specifically expressed on the surface of procyclic and epimastigote forms in the fly. Procyclin genes are already transcribed in bloodstream forms, but stable mRNA, and later the protein, are first detected when the parasites begin to differentiate into procyclic forms. We have now identified a region of 16 bases that forms part of a predicted stem-loop structure in the 3' untranslated regions of different procyclin mRNAs; both the sequence and the secondary structure of this 16-mer appear to be required for efficient translation of a reporter gene in procyclic forms. The level of steady-state mRNA, its polyadenylylation, and its distribution in the cell are all unaffected by the presence or absence of this element. Deletion of the 16-mer alone reduces expression more than removal or reversal of the entire 3' untranslated region and flanking region, suggesting that there are additional negative regulatory elements in the same 3' untranslated region.
p53 binds single-stranded DNA ends and catalyzes DNA renaturation and strand transfer.
Authors: Bakalkin G , Yakovleva T , Selivanova G , Magnusson K P , Szekely L , Kiseleva E , Klein G , Terenius L , Wiman K G
Published: May 2006

The p53 tumor-suppressor protein has previously been shown to bind double-stranded and single-stranded DNA. We report that the p53 protein can bind single-stranded DNA ends and catalyze DNA renaturation and DNA strand transfer. Both a bacterially expressed wild-type p53 protein and a glutathione S-transferase-wild-type p53 fusion protein catalyzed renaturation of different short (25- to 76-nt) complementary single-stranded DNA fragments and promoted strand transfer between short (36-bp) duplex DNA and complementary single-stranded DNA. Mutant p53 fusion proteins carrying amino acid substitutions Glu-213, Ile-237, or Tyr-238, derived from mutant p53 genes of Burkitt lymphomas, failed to catalyze these reactions. Wild-type p53 had significantly higher binding affinity for short (36- to 76-nt) than for longer (> or = 462-nt) single-stranded DNA fragments in an electrophoretic mobility-shift assay. Moreover, electron microscopy showed that p53 preferentially binds single-stranded DNA ends. Binding of DNA ends to p53 oligomers may allow alignment of complementary strands. These findings suggest that p53 may play a direct role in the repair of DNA breaks, including the joining of complementary single-stranded DNA ends.
Protein-tyrosine kinase activity tightly associated with human type II Fc gamma receptors.
Authors: Sármay G , Pecht I , Gergely J
Published: May 2006

Stimulation of B cells by clustering their surface immunoglobulins (sIg) leads to enhanced phosphorylation of several cellular proteins on Ser and Tyr residues. The type II Fc gamma receptor (Fc gamma RII) is one of those proteins that undergo Ser phosphorylation. Upon affinity isolation of the Fc gamma RII, several molecular entities are coisolated from Triton X-100 lysates of BL41 Burkitt lymphoma line which undergo "in vitro" (cell free) phosphorylation in the immune complex-associated kinase assay. Furthermore, several molecules phosphorylated on Tyr upon sIgM cross-linking in the intact cells are coisolated with Fc gamma RII. The 59-kDa coprecipitated component is identified as the protein-tyrosine kinase (PTK) fyn. Clustering the sIgM molecules enhanced the in vitro phosphorylation of all molecules coprecipitated with Fc gamma RII as well as that of the exogenously added PTK substrate, enolase. Kinase renaturation assays suggest that at least two major renaturable protein kinases (59 kDa and 85-90 kDa) associate with Fc gamma RII. Whereas the 59-kDa component comigrates with the PTK fyn, the 85- to 90-kDa one is an unidentified Ser/Thr kinase. These data suggest that Fc gamma RII exists in the B-cell membrane as part of a multimolecular complex including protein kinases, activities of which are regulated by clustering of the antigen receptors.
Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth.
Authors: Janosi L , Shimizu I , Kaji A
Published: May 2006

Ribosome releasing factor, product of the frr gene in Escherichia coli, is responsible for dissociation of ribosomes from mRNA after the termination of translation. It functions to "recycle" ribosomes and is renamed ribosome recycling factor in this paper. An E. coli strain was constructed (MC1061-2), which carried frame-shifted frr in the chromosome and wild-type frr on a temperature-sensitive plasmid. MC1061-2 is temperature-sensitive in its growth and does not segregate its frr-carrying plasmid under the plasmid incompatibility pressure. In contrast, isogenic E. coli carrying wild-type frr in the chromosome and mutated frr on the temperature-sensitive plasmid is not temperature-sensitive in its growth and segregates its plasmid from incompatible plasmids. All spontaneously formed thermoresistant colonies derived from MC1061-2 carried wild-type frr that resided either in the bacterial chromosome by re-exchange or in the plasmid, which became temperature-resistant. These observations establish that frr is an essential gene for cell growth.
Cloning of human basic A1, a distinct 59-kDa dystrophin-associated protein encoded on chromosome 8q23-24.
Authors: Ahn A H , Yoshida M , Anderson M S , Feener C A , Selig S , Hagiwara Y , Ozawa E , Kunkel L M
Published: May 2006

Duchenne and Becker muscular dystrophies are caused by defects of dystrophin, which forms a part of the membrane cytoskeleton of specialized cells such as muscle. It has been previously shown that the dystrophin-associated protein A1 (59-kDa DAP) is actually a heterogeneous group of phosphorylated proteins consisting of an acidic (alpha-A1) and a distinct basic (beta-A1) component. Partial peptide sequence of the A1 complex purified from rabbit muscle permitted the design of oligonucleotide probes that were used to isolate a cDNA for one human isoform of A1. This cDNA encodes a basic A1 isoform that is distinct from the recently described syntrophins in Torpedo and mouse and is expressed in many tissues with at least five distinct mRNA species of 5.9, 4.8, 4.3, 3.1, and 1.5 kb. A comparison of our human cDNA sequence with the GenBank expressed sequence tag (EST) data base has identified a relative from human skeletal muscle, EST25263, which is probably a human homologue of the published mouse syntrophin 2. We have mapped the human basic component of A1 and EST25263 genes to chromosomes 8q23-24 and 16, respectively.
Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane.
Authors: Altenberg G A , Vanoye C G , Horton J K , Reuss L
Published: May 2006

P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is an ATPase thought to actively export cytotoxic drugs. It has been proposed that Pgp transports drugs directly from the lipid bilayer to the external medium ("vacuum cleaner" hypothesis). A possible mechanism for this model is that the Pgp is a flippase--i.e., it catalyzes the translocation of hydrophobic substrates from the inner to the outer leaflet of the cell membrane. Two immediate predictions of the vacuum cleaner and flippase hypotheses are that the apparent unidirectional influx of substrate should be less in Pgp-expressing than in Pgp-lacking cells and that this difference should be abolished by inhibition of the Pgp. We used Chinese hamster fibroblasts with different levels of Pgp expression to measure true unidirectional fluxes of rhodamine 123 (R123), a Pgp-transported fluorescent dye that accumulates in mitochondria (hence, its cytosolic concentration remains low at short times after external addition). The unidirectional efflux of R123 was proportional to the level of Pgp expression and was reduced by Pgp inhibitors. The unidirectional influx of R123 was the same in sensitive and resistant cells--i.e., independent of the level of Pgp expression and insensitive to inhibitors of R123 efflux. From these results, we rule out the vacuum cleaner and flippase hypotheses and conclude that Pgp extracts the actively transported substrates from the cytosol and not from the plasma membrane.
The nonpeptide WIN 64338 is a bradykinin B2 receptor antagonist.
Authors: Sawutz D G , Salvino J M , Dolle R E , Casiano F , Ward S J , Houck W T , Faunce D M , Douty B D , Baizman E , Awad M M
Published: May 2006

We report the synthesis and in vitro biological activity of the nonpeptide bradykinin receptor antagonist WIN 64338, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2- naphthyl)-1-oxopropyl]amino]phenyl]methyl]tributylphosphonium chloride monohydrochloride. WIN 64338 inhibits [3H]-bradykinin binding to the bradykinin B2 receptor on human IMR-90 cells with a binding inhibition constant (Ki) of 64 +/- 8 nM and demonstrates competitive inhibition of bradykinin-stimulated 45Ca2+ efflux from IMR-90 cells (pA2 = 7.1). The antagonist inhibits bradykinin-mediated guinea pig ileum contractility (pA2 = 8.2) and has significantly weaker activity against acetylcholine-induced contractility in the same preparation. WIN 64338 is not active in a rabbit aorta bradykinin B1 receptor assay, demonstrating that it is a selective bradykinin B2 receptor antagonist. The compound inhibits [3H]quinuclidinyl benzilate binding to the rat brain muscarinic receptor (Ki = 350 nM) but is 25- to 100-fold more selective for the bradykinin receptor compared with other receptors against which it has been tested. Synthesis of WIN 64338 has provided a nonpeptide competitive bradykinin B2 antagonist active in both bradykinin radioligand binding and functional assays.
TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR.
Authors: Park H , Davies M V , Langland J O , Chang H W , Nam Y S , Tartaglia J , Paoletti E , Jacobs B L , Kaufman R J , Venkatesan S
Published: May 2006

A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA. The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA of human immunodeficiency virus type 1. TRBP inhibited phosphorylation of the interferon-induced ribosome-associated protein kinase PKR and of the eukaryotic translation initiation factor eIF-2 alpha in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR. TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant lacking the expression of the dsRNA-binding protein E3L. These results implicate TRBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the inhibition of PKR activation and stimulate translation in a localized manner.
High-resolution laser desorption mass spectrometry of peptides and small proteins.
Authors: McIver R T , Li Y , Hunter R L
Published: May 2006

Matrix-assisted laser desorption/ionization (MALDI) has been used with an external ion source Fourier-transform mass spectrometer to obtain the highest mass resolution ever, to our knowledge, demonstrated for laser-produced ions (m/delta m = 1,100,000 for [Arg8]vasopressin, 228,000 for melittin, and 90,000 for bovine insulin). The peaks in the isotope cluster for bovine insulin are fully resolved, and the mass measurement accuracy is an order of magnitude better than can be achieved with time-of-flight mass spectrometry. With the method described here, analyte is applied to a sample probe and mixed with a solution containing a matrix material (2,5-dihydroxybenzoic acid) that strongly absorbs ultraviolet light. Upon irradiation with a pulse from an excimer laser (353 nm, 2 mJ), a large number of intact protonated molecular ions are produced. The ions are focused by a 117-cm-long quadrupole ion guide and injected into an ion cyclotron resonance analyzer cell located inside the bore of a 6.5-T superconducting magnet. A pulse of argon buffer gas cools the ions prior to detection. One of the principal advantages of an external ion source Fourier-transform mass spectrometer is that the ion formation and ion detection processes are separated and can be independently optimized.
Amino acid receptors of midget and parasol ganglion cells in primate retina.
Authors: Zhou Z J , Marshak D W , Fain G L
Published: May 2006

Primate retinas contain two major ganglion cell types. Midget (or P type) cells have relatively sustained responses to light; the amplitude and polarity of these responses vary with stimulus wavelength. Parasol (or M type) cells are more sensitive to stimulus contrast and respond more transiently but are not selective for color. Both types can be further subdivided into a and b subtypes, according to the level of their dendritic stratification in the inner plexiform layer. To determine whether differences in receptors for amino acid transmitters are the basis for any differences in ganglion cell light responses, we made whole-cell, patch-clamp recordings from identified ganglion cells in slice preparations of macaque and baboon retinas. We found that midget and parasol cells of both a and b types had similar responses to excitatory amino acids, including kainate, alpha-amino-3-hydroxy-5-methylisoxalzole-4-propionic acid, and N-methyl-D-aspartate, with reversal potentials near the equilibrium potential for cations. Kainate responses were blocked by 6,7-dinitroquinoxaline, and N-methyl-D-aspartate responses were blocked by D-(-)-2-amino-7-phosphonoheptanoic acid. The four types of ganglion cells also had similar responses to bath-applied inhibitory amino acids. All cells had both gamma-aminobutyric acid and glycine receptors with reversal potentials near the equilibrium potential for Cl-, and the relative amplitudes of the responses to excitatory and inhibitory amino acids were similar among the various cell types. These results suggest that the differences in response properties of the different classes of ganglion cells in primate retina may be determined, to a significant degree, by the properties of the amacrine and bipolar cells that provide their input rather than by the nature of their postsynaptic receptors.
Fas and its ligand in a general mechanism of T-cell-mediated cytotoxicity.
Authors: Hanabuchi S , Koyanagi M , Kawasaki A , Shinohara N , Matsuzawa A , Nishimura Y , Kobayashi Y , Yonehara S , Yagita H , Okumura K
Published: May 2006

To investigate the mechanisms of T-cell-mediated cytotoxicity, we estimated the involvement of apoptosis-inducing Fas molecule on the target cells and its ligand on the effector cells. When redirected by ConA or anti-CD3 monoclonal antibody, a CD4+ T-cell clone, BK1, could lyse the target cells expressing wild-type Fas molecule but not those expressing death signaling-deficient mutants. This indicates the involvement of Fas-mediated signal transduction in the target cell lysis by BK1. Anti-CD3-activated but not resting BK1 expressed Fas ligand as detected by binding of a soluble Fas-Ig fusion protein, and the BK1-mediated cytotoxicity was blocked by the addition of Fas-Ig, implicating the inducible Fas ligand in the BK1 cytotoxicity. Ability to exert the Fas-mediated cytotoxicity was not confined to BK1, but splenic CD4+ T cells and, to a lesser extent, CD8+ T cells could also exert the Fas-dependent target cell lysis. This indicates that the Fas-mediated target cell lytic pathway can be generally involved in the T-cell-mediated cytotoxicity. Interestingly, CD4+ T cells prepared from gld/gld mice did not mediate the Fas-mediated cytotoxicity, indicating defective expression of functional Fas ligand in gld mice.
The interleukin (IL) 2 receptor beta chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.
Authors: Bamford R N , Grant A J , Burton J D , Peters C , Kurys G , Goldman C K , Brennan J , Roessler E , Waldmann T A
Published: May 2006

Late-phase human T-cell lymphotropic virus I-associated adult T-cell leukemia cells express IL-2 receptors (IL-2R) but no longer produce IL-2. We have reported that the IL-2-independent adult T-cell leukemia line HuT-102 secretes a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and lymphokine-activated killer cell activity. Stimulation of proliferation of the cytokine-dependent human T-cell line Kit-225 mediated by HuT-102-conditioned medium or by 3200-fold-purified IL-T was not blocked by the addition of antibodies against IL-2 or IL-2R alpha subunit. However, IL-T-mediated stimulation of this human T-cell line was inhibited by addition of Mik-beta 1, an antibody that binds specifically to IL-2R beta subunit. In addition, the activation of large granular lymphocytes to lymphokine-activated killer cells mediated by IL-T-containing conditioned medium was not blocked by antibodies directed toward IL-2 or IL-2 alpha but was inhibited by an antibody to IL-2R beta, suggesting the requirement of this receptor subunit for IL-T action. This conclusion was confirmed using an IL-3-dependent murine myeloid precursor cell line, 32D, that expresses IL-2R alpha and IL-2R gamma, but not IL-2R beta. Neither IL-2 nor IL-T stimulated 32D cell proliferation. However, after transfection with the gene encoding human IL-2R beta, 32D beta cells proliferated on addition of either cytokine. The IL-T-mediated stimulation of 32D beta proliferation was inhibited by an anti-IL-2R beta antibody but not by an anti-IL-2 antibody. Thus, the IL-T-mediated stimulation of T-cell and lymphokine-activated killer cell activation requires the expression of the IL-2R beta subunit.