bluuebunny/crossref_metadata_2025 huggingface dataset:
Excision repair at individual bases of the Escherichia coli lacI gene: relation to mutation hot spots and transcription coupling activity.
Authors: Kunala S , Brash D E
Published: May 2006

To determine whether base-to-base variations in the rate of excision repair influence the distribution of mutations, we have developed a method to measure UV photoproducts at individual nucleotides in the Escherichia coli chromosome. Specific gene fragments are 3' end-labeled using a sequence-specific oligonucleotide to direct the site of labeling, and photoproducts are identified by enzymatic incision. On the nontranscribed strand of the E. coli lacI gene, the cyclobutane pyrimidine dimer frequency was 2- to 8-fold higher in chromosomal DNA than in a cloned DNA fragment. The chromosomal lesion frequency corresponded to the frequency of UV-induced mutations at mutation hot spots reported in the literature. Only 0-30% of cyclobutane dimers at various sites on this strand were excised in 20 min. In contrast, repair on the transcribed strand was 80-90% complete in 20 min. However, the transcribed strand contained an excision repair "slow spot" at the site of its single mutation hot spot: At this site, no repair occurred for the first 10 min, after which repair proceeded more slowly than typical of that strand. In an mfd strain, deficient in a factor that couples repair to transcription in cell extracts, the excision rate at individual nucleotides on the transcribed strand was minimal at most sites for at least 30 min. Wild-type E. coli's bias for producing mutations at photoproducts on the nontranscribed strand, reported to require the mfd gene, therefore appears to be due to an excision repair system specific for the transcribed strand of chromosomal DNA.
Stimulatory and inhibitory regulation of calcium-activated potassium channels by guanine nucleotide-binding proteins.
Authors: Kume H , Graziano M P , Kotlikoff M I
Published: May 2006

The regulation of membrane ion channels by guanine nucleotide-binding proteins (G proteins) has been described in numerous tissues. This regulation has been shown to involve the membrane-delimited stimulatory action of G proteins on ion channels. We now show that single calcium-activated potassium channels (KCa channels) in airway smooth muscle cells are both stimulated and inhibited by G proteins in membrane patches. We demonstrate that the beta-adrenergic agonist isoproterenol stimulates channel activity via the alpha subunit of the stimulatory G protein of adenylyl cyclase, Gs, and that channel opening is inhibited by the action of the muscarinic agonist methacholine, acting via a pertussis toxin-sensitive G protein. Isoproterenol stimulated and methacholine inhibited channel activity in the same outside-out patches when GTP was present at the cytosolic surface of the patch. In inside-out patches, addition of GTP and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) augmented channel activity when isoproterenol was included in the patch pipette, and inhibited channel activity when methacholine was included in the pipette. Consistent with these results, in the presence of GTP[gamma S], the alpha subunit of Gs (alpha s.GTP[gamma S] complex) opened KCa channels in a dose-dependent manner, whereas in the presence of guanosine 5'-[beta-thio]diphosphate, alpha s had no effect. By contrast, application of activated alpha i or alpha o proteins did not inhibit channel activity in inside-out patches, indicating that channel inhibition is more complex than a simple alpha subunit/channel interaction, similar to the complex inhibitory regulation of adenylyl cyclase. These results suggest that hormonal regulation of KCa channels shares substantial features with the regulation of adenylyl cyclase and demonstrate that a single ion channel may serve as the regulatory target for the membrane-delimited action of stimulatory and inhibitory G proteins. Moreover, they demonstrate a potentially important functional pathway by which beta-adrenergic and other Gs-linked receptors stimulate relaxation of smooth muscle, independent of cAMP-dependent protein phosphorylation.
Fibrinogen blocks the autoactivation and thrombin-mediated activation of factor XI on dextran sulfate.
Authors: Scott C F , Colman R W
Published: May 2006

The intrinsic pathway of blood coagulation is activated when factor XIa, one of the three contact-system enzymes, is generated and then activates factor IX. Factor XI has been shown to be efficiently activated in vitro by surface-bound factor XIIa after factor XI is transported to the surface by its cofactor, high molecular weight kininogen (HK). However, individuals lacking any of the three contact-system proteins--namely, factor XII, prekallikrein, and HK--do not suffer from bleeding abnormalities. This mystery has led several investigators to search for an "alternate" activation pathway for factor XI. Recently, factor XI has been reported to be autoactivated on the soluble "surface" dextran sulfate, and thrombin was shown to accelerate the autoactivation. However, it was also reported that HK, the cofactor for factor XIIa-mediated activation of factor XI, actually diminishes the thrombin-catalyzed activation rate of factor XI. Nonetheless, it was suggested that thrombin was a more efficient activator than factor XIIa. In this report we investigated the effect of fibrinogen, the major coagulation protein in plasma, on the activation rate of factor XI. Fibrinogen, the preferred substrate for thrombin in plasma, virtually prevented autoactivation of factor XI as well as the thrombin-mediated activation of factor XI, while having no effect on factor XIIa-catalyzed activation. HK dramatically curtailed the autoactivation of factor XI in addition to the thrombin-mediated activation. These data indicate that factor XI would not be autoactivated in a plasma environment, and thrombin would, therefore, be unlikely to potentiate the activation. We believe that the "missing pathway" for factor XI activation remains an enigma that warrants further investigation.
Binding and transport of gangliosides by prosaposin.
Authors: Hiraiwa M , Soeda S , Kishimoto Y , O'Brien J S
Published: May 2006

Prosaposin, the precursor of saposins A, B, C, and D, which activate lysosomal hydrolysis of sphingolipids, exists in various tissues and body fluids and is especially abundant in the nervous system. Prosaposin and saposins A,B, C, and D formed stable complexes with 13 different gangliosides as measured by an assay using column chromatography. Gangliosides of the gangliotetraose type (a series) were bound with high affinity, whereas b series gangliosides, O-acetylated gangliosides, and gangliosides with shorter carbohydrate chains, were bound with lower affinity. Prosaposin and saposins transferred gangliosides from donor liposomes to erythrocyte ghost membranes. Prosaposin also stimulated ganglioside GM1 beta-galactosidase more than mature saposins. Prosaposin exists as a secretory protein and as an integral membrane protein, and we propose that prosaposin is active as a ganglioside binding and transport protein in vivo.
Carbohydrate cycling in signal transduction: parafusin, a phosphoglycoprotein and possible Ca(2+)-dependent transducer molecule in exocytosis in Paramecium.
Authors: Subramanian S V , Satir B H
Published: May 2006

Parafusin, a cytosolic phosphoglycoprotein of M(r) 63,000, is dephosphorylated and rephosphorylated rapidly in a Ca(2+)-dependent manner upon stimulation of exocytosis in vivo in wild-type (wt) Paramecium. In contrast, the temperature-sensitive exocytosis mutant nd9, grown at the nonpermissive temperature (27 degrees C), does not exocytose or dephosphorylate parafusin upon stimulation in the presence of Ca2+; grown at the permissive temperature (18 degrees C), nd9 cells show a wt phenotype. Parafusin contains two types of phosphorylation sites: one where glucose 1-phosphate is added by an alpha-glucose-1-phosphate phosphotransferase and removed by a phosphodiesterase and one where phosphate from ATP is added directly to a serine residue by a protein kinase and removed by a phosphatase. We show here that, in cell fractions from wt Paramecium, both reactions can be carried out in vitro by using uridine(5'-[beta-[35S]thio])diphospho(1)-glucose (UDP[beta 35S]-Glc) and [gamma-32P]ATP, respectively. The characteristics of these pathways are different. Specifically, in the presence of Ca2+, the amount of UDP[beta 35S]-Glc label in parafusin is reduced. In contrast, identical labeling experiments with [gamma-32P]ATP show that Ca2+ enhances labeling of parafusin. Mg2+ had no appreciable effect on either labeling. Removal of the UDP[beta 35S]-Glc label on parafusin in the presence of Ca2+ correlates with the in vivo dephosphorylation seen upon exocytosis. Incubations with UDP[beta 35S]-Glc were then performed with homogenates and nd9 cell fractions grown at 27 degrees C under the ionic conditions used for wt cells. These labelings were not affected by Ca2+, in contrast to results from wt cells but in accord with those obtained earlier with nd9-27 mutant cells in vivo. Factors responsible for both dephosphorylation and Ca2+ sensitivity were found in the high-speed pellet (P2) in wt cells, suggesting that the putative phosphodiesterase is in this fraction and that the defect in the mutant nd9-27 residues in the Ca2+ activation of the phosphodiesterase. We conclude that the in vivo dephosphorylation of parafusin that occurs upon exocytosis is a dephosphoglucosylation due to removal of the alpha-glucose 1-phosphate and more generally that carbohydrates on cytoplasmic glycoproteins may be cyclically added and/or removed in response to extracellular stimuli.
Spermine and spermidine protection of plasmid DNA against single-strand breaks induced by singlet oxygen.
Authors: Khan A U , Di Mascio P , Medeiros M H , Wilson T
Published: May 2006

Oxidative damage to DNA induced by singlet molecular oxygen (1O2*) includes single-strand breaks, which the biologically occurring 1O2* quenchers spermine and spermidine are shown to prevent. These polyamines at a physiological concentration (10 mM) reduce the percentage of the open circular form of pBR322 plasmid DNA, which is generated at the expense of the native supercoiled form when the plasmids are incubated with a chemical source of 1O2*, the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate. Spermine and spermidine can be expected to protect DNA against other damaging effects of 1O2*.
Interaction of the yeast Swi4 and Swi6 cell cycle regulatory proteins in vitro.
Authors: Andrews B J , Moore L A
Published: May 2006

In budding yeast, two transcription factors, Swi4 and Swi6, control the expression of important cell cycle regulatory proteins (the G1 cyclins, Cln1 and Cln2, and the cyclin-like Hcs26) as well as the HO gene, whose product initiates mating-type switching. Both Swi4 and Swi6 are components of a protein complex that forms at a repeated sequence element, SCB (SWI4, -6-dependent cell cycle box), found in the upstream regulatory sequences of target genes. We show, by using proteins synthesized in vitro, a direct association between Swi4 and Swi6. The cdc10-Swi6 or ankyrin motifs present in both Swi4 and Swi6 are dispensable for their association, which is mediated instead by a region near the C terminus of each protein. Furthermore, we show that interaction with Swi6 is not necessary for specific recognition of the SCB sequence by the Swi4 protein; we propose that Swi4 is responsible for binding to the SCB sequence while Swi6, through its association with Swi4, regulates activity of the complex.
Helix-loop-helix transcription factors E12 and E47 are not essential for skeletal or cardiac myogenesis, erythropoiesis, chondrogenesis, or neurogenesis.
Authors: Zhuang Y , Kim C G , Bartelmez S , Cheng P , Groudine M , Weintraub H
Published: May 2006

E12 and E47 are two non-tissue-specific helix-loop-helix (HLH) transcription factors encoded by the E2A gene. Previous studies suggested that they are involved in regulation of differentiation in many tissue types including muscle, blood, and nerve through direct heterodimer interactions with tissue-specific HLH proteins. To gain further genetic insight into the functions of E12 and E47 during cell differentiation, we mutated both copies of the E2A gene in mouse embryonic stem (ES) cells and then tested the effect on differentiation in vitro. We find that the ES cells lacking functional E12 and E47 are capable of differentiating into both skeletal and cardiac muscle, erythrocytes, neurons, and cartilage that the same extent as wild-type cells. These results indicate that the E2A gene is not essential for differentiation of these cell types and suggest that redundant genes may control these developmental pathways.
Magic numbers for metallic clusters and the principle of maximum hardness.
Authors: Harbola M K
Published: May 2006

It is shown that for relatively more stable metallic clusters (those with magic number of atoms) the chemical hardness (I-A) too is relatively larger. Thus the occurrence of magic numbers for metal clusters whose stability is determined by their electronic shell structure can be understood as a manifestation of the principle of maximum hardness. This may also represent a possible way of delineating clusters with stability dominated by their electronic shell structure from those for which the magic numbers occur as a result of their geometric structure.
Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing.
Authors: Munroe S H , Dong X F
Published: May 2006

Within the nucleus, pre-mRNA molecules are complexed with a set of proteins to form heterogeneous nuclear ribonucleoprotein complexes. A1, an abundant RNA binding protein present in these complexes, has been shown to bind selectively to single-stranded RNAs and destabilize base-pairing interactions. In this study.A1 is shown to promote the rate of annealing of complementary RNA strands greater than 300-fold under a wide range of salt concentration and temperature. Maximal annealing is observed under saturating or near saturating concentrations of protein, but annealing decreases sharply at both higher and lower concentrations of A1. Kinetic analysis shows that the rate of annealing is not strictly first or second order with respect to RNA at a ratio of protein/RNA that gives optimal rates of annealing. This result suggests that A1 protein may affect more than one step in the annealing reaction. Two polypeptides representing different domains of A1 were also examined for annealing activity. UP1, a proteolytic fragment that represents the N-terminal two-thirds of A1, displays very limited annealing activity. In contrast, a peptide consisting of 48 amino acid residues from the glycine-rich C-terminal region promotes annealing at a rate almost one-quarter that observed with intact A1. The RNA.RNA annealing activity of A1 may play a role in pre-mRNA splicing and other aspects of nuclear mRNA metabolism.
Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling.
Authors: Kliewer S A , Umesono K , Heyman R A , Mangelsdorf D J , Dyck J A , Evans R M
Published: May 2006

We have recently described the properties of direct repeats (DRs) of the half-site AGGTCA as hormone response elements (HREs). According to our results, spacing the half sites by 3, 4, or 5 nucleotides determines specificity of response for vitamin D3, thyroid hormone, and retinoic acid receptors, respectively. This so-called 3-4-5 rule led to the prediction that remaining spacing options of 0, 1, and 2 might serve as targets for other nuclear receptors. A concurrent prediction is that receptors recognizing common sites might display more complex or combinatorial interactions. In exploring these predictions, we discovered that both the retinoid X receptor (RXR) and COUP-TF bind preferentially to a DR-1 motif. In vivo, RXR and COUP-TF display antagonistic action such that RXR-mediated activation is fully repressed by COUP-TF. In vitro studies reveal that COUP-TF and RXR form heterodimers on DR-1. Thus, these results support a general proposal in which the half-site spacing preferences may be used as a means to decipher potentially complex and interactive regulatory circuits.
Highly conserved repetitive DNA sequences are present at human centromeres.
Authors: Grady D L , Ratliff R L , Robinson D L , McCanlies E C , Meyne J , Moyzis R K
Published: May 2006

Highly conserved repetitive DNA sequence clones, largely consisting of (GGAAT)n repeats, have been isolated from a human recombinant repetitive DNA library by high-stringency hybridization with rodent repetitive DNA. This sequence, the predominant repetitive sequence in human satellites II and III, is similar to the essential core DNA of the Saccharomyces cerevisiae centromere, centromere DNA element (CDE) III. In situ hybridization to human telophase and Drosophila polytene chromosomes shows localization of the (GGAAT)n sequence to centromeric regions. Hyperchromicity studies indicate that the (GGAAT)n sequence exhibits unusual hydrogen bonding properties. The purine-rich strand alone has the same thermal stability as the duplex. Hyperchromicity studies of synthetic DNA variants indicate that all sequences with the composition (AATGN)n exhibit this unusual thermal stability. DNA-mobility-shift assays indicate that specific HeLa-cell nuclear proteins recognize this sequence with a relative affinity greater than 10(5). The extreme evolutionary conservation of this DNA sequence, its centromeric location, its unusual hydrogen bonding properties, its high affinity for specific nuclear proteins, and its similarity to functional centromeres isolated from yeast suggest that this sequence may be a component of the functional human centromere.
Rational design of substituted tripyrrole peptides that complex with DNA by both selective minor-groove binding and electrostatic interaction with the phosphate backbone.
Authors: Bruice T C , Mei H Y , He G X , Lopez V
Published: May 2006

The structures of the compounds we call 3a, 3b, and 3c-compounds that incorporate (i) the tripyrrole peptide of the minor-groove-binding distamycin class of compounds and (ii) polyamine ligands that extend from the minor groove and can interact with phosphodiester bonds--were arrived at by computer-graphics designing by using the x-ray structure of distamycin A complexed in the minor groove of d(CGCAAATTTGCG)2. Compounds 3a, 3b, and 3c are elaborations of distamycin analog 2, designed for improved stability in solution and easier synthesis and purification, which itself binds weakly to DNA. Compounds 3a, 3b, and 3c have been synthesized, and the interaction of distamycin A, 2, 3a, 3b, and 3c with calf thymus DNA, poly(dA-dT), poly(dG-dC), poly(dI-dC), pBR322 superhelical plasmid DNA, and, in the case of 3b, T4 coliphage DNA have been studied. The following pertinent conclusions can be drawn. Binding of 3a, 3b, and 3c occurs in the minor groove of DNA and, because of favorable electrostatic interaction of diprotonated polyamine side chains and DNA phosphodiester linkages, the tenacity of DNA binding and site specificity of 3a, 3b, and 3c are comparable to that of native distamycin A. 3b has been found to induce changes in the superhelical density of pBR322 plasmid DNA. The study establishes that the central pyrrole N-CH3 substituent of 2 can be replaced by bulky polyamine metal ligands to create any number of compounds that bind into the minor groove at A + T-rich sites and are putative catalysts for the hydrolysis of DNA.
The retinoblastoma-susceptibility gene product becomes phosphorylated in multiple stages during cell cycle entry and progression.
Authors: DeCaprio J A , Furukawa Y , Ajchenbaum F , Griffin J D , Livingston D M
Published: May 2006

The retinoblastoma-susceptibility gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. We characterized RB phosphorylation after mitogenic stimulation of primary human T lymphocytes, initially arrested in the G0 state. RB is phosphorylated in at least three steps when T cells are driven into the cell cycle. The first event occurs during mid G1 phase, the second during S phase, and the third in G2/M. Tryptic phosphopeptide mapping indicates that the different phosphorylation events occur, at least in part, on different residues in RB. Given the known relationship of the RB phosphorylation state to function, it is possible that RB regulates growth at multiple points in the cell cycle.
Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT1-1 GnRH neuronal cell line.
Authors: Martínez de la Escalera G , Choi A L , Weiner R I
Published: May 2006

The immortalized neuronal cell line GT1-1 was used to investigate the endogenous pattern of GnRH release. The GT1-1 cell line was derived from a GnRH-secreting tumor in a transgenic mouse induced by genetically targeted expression of the potent simian virus 40 oncogene encoding tumor antigen. Cells attached to coverslips were superfused in Sykes-Moore chambers with Locke's medium, Ca(2+)-free Locke's medium, or Opti-MEM (another defined medium) for 2 hr, and samples were collected at 4-min intervals. Release of GnRH in 17 of 18 superfusion chambers was seen to be pulsatile when data were analyzed by cluster analysis. No significant differences were observed whether only one or both of the coverslips forming the chamber were coated with cells. Pulses exhibited a mean interpulse interval of 25.8 +/- 1.5 min, a mean duration of 18.8 +/- 1.4 min, and a mean amplitude of 150.5 +/- 6.0% above preceding nadir. The removal of Ca2+ from the Locke's medium resulted in the progressive reduction of the amplitude and eventually in the absence of identifiable pulses. Pulses reappeared after the return of Ca2+ to the medium. It is concluded that the GT1-1 cell line secretes GnRH in a rhythmic pattern. These findings suggest that the pulsatile release of GnRH (GnRH pulse generator) may be an intrinsic characteristic of the GnRH neurons. Synchronization of pulsatile release from individual neurons could be mediated via numerous cell-to-cell contacts observed in the cultured cells on coverslips. Synchronization of GnRH release from cells on two physically separated coverslips forming a chamber would appear to be accomplished by a diffusible mediator.
The dual-mode quaternary structure of seminal RNase.
Authors: Piccoli R , Tamburrini M , Piccialli G , Di Donato A , Parente A , D'Alessio G
Published: May 2006

Bovine seminal ribonuclease, the only dimeric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially transformed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells.
Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes.
Authors: Rousset F , Garcia E , Defrance T , Péronne C , Vezzio N , Hsu D H , Kastelein R , Moore K W , Banchereau J
Published: May 2006

Interleukin 10 (IL-10), originally identified as a TH2 helper T-cell product able to inhibit cytokine production by TH1 cells, is highly homologous to BCRF1 (viral IL-10), an open reading frame in the Epstein-Barr virus genome. Here, we show that human and viral IL-10 stimulate DNA replication of B lymphocytes activated either via their antigen receptor or via their CD40 antigen. IL-4 and IL-10 display additive effects and induce a strong increase in the number of viable cells. Moreover, IL-10 induces activated B cells to secrete large amounts of IgG, IgA, and IgM, and the combination of IL-10 and IL-4 results in the secretion of the four immunoglobulin isotypes. Thus, IL-10 may play an important role in the amplification of humoral responses.
Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate.
Authors: Yeh P , Landais D , Lemaître M , Maury I , Crenne J Y , Becquart J , Murry-Brelier A , Boucher F , Montay G , Fleer R
Published: May 2006

Due to its remarkably long half-life, together with its wide in vivo distribution and its lack of enzymatic or immunological functions, human serum albumin (HSA) represents an optimal carrier for therapeutic peptides/proteins aimed at interacting with cellular or molecular components of the vascular and interstitial compartments. As an example, we designed a genetically engineered HSA-CD4 hybrid aimed at specifically blocking the entry of the human immunodeficiency virus into CD4+ cells. In contrast with CD4, HSA-CD4 is correctly processed and efficiently secreted by Kluyveromyces yeasts. In addition, its CD4 moiety exhibits binding and antiviral in vitro properties similar to those of soluble CD4. Finally, the elimination half-life of HSA-CD4 in a rabbit experimental model is comparable to that of control HSA and 140-fold higher than that of soluble CD4. These results indicate that the genetic fusion of bioactive peptides to HSA is a plausible approach toward the design and recovery of secreted therapeutic HSA derivatives with appropriate pharmacokinetic properties.
Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability.
Authors: Fraser C C , Szilvassy S J , Eaves C J , Humphries R K
Published: May 2006

Marrow cells from male mice pretreated with 5-fluorouracil were infected with helper-free neomycin-resistant (neor) recombinant retrovirus and then used to initiate long-term cultures (LTC) on irradiated adherent marrow feeder layers. Four weeks later LTC cells were harvested and injected into lethally irradiated female recipients either alone or together with 2 x 10(5) female marrow cells with selectively compromised long-term repopulating potential to assay for totipotent and competitive repopulating units (CRU), respectively. A total of 46 unique clones were detected in recipients 5 wk to 7 mo after transplant. Half of these clones (22 of 46) included both lymphoid and myeloid progeny. Eight of the 22 lympho-myeloid clones were represented in multiple recipients, in some cases after injection of limiting numbers of CRU, thus indicating repopulation from sibling totipotent stem cells generated during the initial 4-wk period in LTC. Serial analysis of cells released into the nonadherent fraction of LTC for up to 7 wk provided additional evidence of the continuing proliferation in LTC of totipotent stem cells with long-term repopulating potential. The frequency of CRU determined from limiting-dilution analyses of LTC-derived cells was the same for recipients analyzed at 5 wk or 7 mo after transplantation and was also the same whether marrow or thymus repopulation was assessed. These assays showed that concurrent with the expansion of some totipotent cells revealed by retroviral marking, there was a slow but net 6.5-fold decrease in total CRU numbers after 4 wk in LTC. These results show the capacity of some totipotent hematopoietic stem cells to be maintained and amplified over extensive time periods in vitro without diminution of their long-term in vivo repopulating potential. These results also set the stage for analogous studies of human stem cell selection and expansion in vitro, which may be important for future gene therapy protocols.
Evidence that the ras oncogene-encoded p21 protein induces oocyte maturation via activation of protein kinase C.
Authors: Chung D L , Brandt-Rauf P W , Weinstein I B , Nishimura S , Yamaizumi Z , Murphy R B , Pincus M R
Published: May 2006

The ras oncogene-encoded p21 protein is known to induce cell maturation of Xenopus laevis oocytes and malignant transformation of NIH 3T3 mouse fibroblasts. The pathways involved in oocytes and NIH 3T3 cells appear to be similar to one another. For example, in both cases, the ras p21-induced cellular events involve increased intracellular levels of the second messengers diacylglycerol and inositol phosphates, the former of which activates protein kinase C (PKC). To investigate the pathway of ras-induced oocyte maturation, we have explored the relationship between p21 protein and PKC. We show that the maturation signal from oncogenic p21 microinjected into Xenopus oocytes is completely blocked by the relatively specific PKC inhibitor CGP 41251, a staurosporine analogue that selectively inhibits PKC, but not by an inactive analogue of staurosporine, CGP 42700. Microinjection of purified PKC or of phorbol ester induces maturation of oocytes. PKC-induced maturation is inhibited by CGP 41251 but not by CGP 42700. Maturation induced by microinjected PKC is also not inhibited by two specific anti-p21 agents, the inactivating anti-p21 monoclonal antibody Y13-259 and the amino acid derivative azatyrosine. Both of these agents block p21-induced cell maturation. These results suggest that ras effects depend upon the action of PKC, whose activation is an event that occurs downstream of p21 in the maturation signal pathway.
Activation of the cytotactin promoter by the homeobox-containing gene Evx-1.
Authors: Jones F S , Chalepakis G , Gruss P , Edelman G M
Published: May 2006

Cytotactin is a morphoregulatory molecule of the extracellular matrix affecting cell shape, division, and migration that appears in a characteristic and complex site-restricted pattern during embryogenesis. The promoter region of the gene that encodes chicken cytotactin contains a variety of potential regulatory sequences. These include putative binding sites for homeodomain proteins and a phorbol 12-O-tetradecanoate 13-acetate response element (TRE)/AP-1 element, a potential target for transcription factors thought to be involved in growth-factor signal transduction. To determine the effects of homeobox-containing genes on cytotactin promoter activity, we conducted a series of cotransfection experiments on NIH 3T3 cells using cytotactin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs and plasmids driving the expression of mouse homeobox genes Evx-1 and Hox-1.3. cotransfection with Evx-1 stimulated cytotactin promoter activity whereas cotransfection in control experiments with Hox-1.3 had no effect. To localize the sequences required for Evx-1 activation, we tested a series of deletions in the cytotactin promoter. An 89-base-pair region containing a consensus TRE/AP-1 element was found to be required for activation. An oligonucleotide segment containing this TRE/AP-1 site was found to confer Evx-1 inducibility on a simian virus 40 minimal promoter; mutation of the TRE/AP-1 site abolished this activity. To explore the potential role of growth factors in cytotactin promoter activation, chicken embryo fibroblasts, which are known to synthesize cytotactin, were first transfected with cytotactin promoter constructs and cultured under minimal conditions in 1% fetal bovine serum. Although the cells exhibited only low levels of CAT activity under these conditions, cells exposed for 12 h to 10% (vol/vol) fetal bovine serum showed a marked increase in CAT activity. Cotransfection with Evx-1 and cytotactin promoter constructs of cells cultured in 1% fetal bovine serum was sufficient, however, to produce high levels of CAT activity. These findings are consistent with the hypothesis that Evx-1, a homeobox-containing gene, may activate the cytotactin promoter by a mechanism involving a growth-factor signal transduction pathway. More generally, the results support the hypothesis that the place-dependent expression of morphoregulatory molecules may depend upon local cues provided by homeobox genes and their encoded proteins.
Molecular polymorphism and epidemiology of Neisseria meningitidis immunoglobulin A1 proteases.
Authors: Lomholt H , Poulsen K , Caugant D A , Kilian M
Published: May 2006

Neisseria meningitidis is one of several important bacterial pathogens that secrete a specific protease capable of cleaving human immunoglobulin A1 (IgA1) in the hinge region. To obtain further information on this putative virulence factor, we examined the IgA1 protease and iga gene region of 133 isolates of N. meningitidis assigned to 88 multilocus enzyme genotypes and representing major epidemics and carrier strains from 19 countries. Of the two IgA1 cleavage specificities previously observed, isolates associated with epidemics of meningococcal disease showed exclusively type 1 IgA1 protease activity. Considerable heterogeneity of the N. meningitidis IgA1 protease was demonstrated at both the protein and gene levels. Thus, five different forms of IgA1 protease were detected with enzyme-neutralizing antibodies raised in rabbits. An antiserum raised against a single type 2 IgA1 protease inhibited the enzyme activity of all strains examined, a finding of potential significance for the possible application of IgA1 protease in a vaccine against meningococcal disease. Examination of the iga gene region with restriction endonucleases revealed a high degree of polymorphism among strains belonging to some multilocus enzyme genotypes. The different iga gene types did not correlate with cleavage type or inhibition of the IgA1 protease. Our findings indicate that horizontal genetic exchange occurs in vivo with considerably different frequency in different clones of meningococci.
Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver.
Authors: Xu F , Quandt K S , Hultquist D E
Published: May 2006

An NADPH-dependent reductase, first shown in the 1930s to catalyze the methylene blue-dependent reduction of methemoglobin in erythrocytes, has now been characterized as a high-affinity heme-binding protein and has been detected in liver. Highly purified bovine erythrocyte reductase binds protohemin to form a 1:1 complex with a Kd of 7 nM. Binding of protohemin completely inhibits reductase activity. Other tetrapyrroles and fatty acids also bind to the reductase and inhibit its activity. Protoporphyrin, hematoporphyrin, and coproporphyrin form 1:1 complexes with Kd values ranging from 1 to 5 microM. The inhibition constants for a number of saturated and unsaturated fatty acids range from 6 to 52 microM. A protein that is immunologically cross-reactive to the reductase has been detected in the cytosolic fractions of bovine and rat liver and of bovine, rat, rabbit, and human erythrocytes. By immunoblot analysis, the bovine liver and erythrocyte proteins appear identical in size, as do the rat liver and erythrocyte proteins. The concentration of the protein in bovine erythrocytes has been estimated by quantitative immunoblotting to be 10 microM. The detection of this protein in liver cells, the demonstration of its binding properties, and its weak reductase activity bring into question the long-held belief that this is uniquely an erythrocyte protein and that it functions as a reductase.
Molecular surface recognition: determination of geometric fit between proteins and their ligands by correlation techniques.
Authors: Katchalski-Katzir E , Shariv I , Eisenstein M , Friesem A A , Aflalo C , Vakser I A
Published: May 2006

A geometric recognition algorithm was developed to identify molecular surface complementarity. It is based on a purely geometric approach and takes advantage of techniques applied in the field of pattern recognition. The algorithm involves an automated procedure including (i) a digital representation of the molecules (derived from atomic coordinates) by three-dimensional discrete functions that distinguishes between the surface and the interior; (ii) the calculation, using Fourier transformation, of a correlation function that assesses the degree of molecular surface overlap and penetration upon relative shifts of the molecules in three dimensions; and (iii) a scan of the relative orientations of the molecules in three dimensions. The algorithm provides a list of correlation values indicating the extent of geometric match between the surfaces of the molecules; each of these values is associated with six numbers describing the relative position (translation and rotation) of the molecules. The procedure is thus equivalent to a six-dimensional search but much faster by design, and the computation time is only moderately dependent on molecular size. The procedure was tested and validated by using five known complexes for which the correct relative position of the molecules in the respective adducts was successfully predicted. The molecular pairs were deoxyhemoglobin and methemoglobin, tRNA synthetase-tyrosinyl adenylate, aspartic proteinase-peptide inhibitor, and trypsin-trypsin inhibitor. A more realistic test was performed with the last two pairs by using the structures of uncomplexed aspartic proteinase and trypsin inhibitor, respectively. The results are indicative of the extent of conformational changes in the molecules tolerated by the algorithm.
Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions to DNA caused by binding of the anticancer agent cisplatin.
Authors: Bruhn S L , Pil P M , Essigmann J M , Housman D E , Lippard S J
Published: May 2006

Human cDNA clones encoding a structure-specific recognition protein, SSRP1, that binds specifically to DNA modified with cisplatin have been isolated and characterized. The SSRP1 gene maps to human chromosome 11q12. The cDNA clones, obtained by using partial-length cDNAs described previously, predict an 81-kDa protein containing several highly charged domains and a stretch of 75 amino acids 47% identical to a portion of the high mobility group (HMG) protein HMG1. This HMG box most likely constitutes the structure recognition element for cisplatin-modified DNA, with the probable recognition motif being the local duplex unwinding and bending toward the major groove that occurs upon formation of intrastrand cis-[Pt(NH3)2]2+ d(GpG) and d(ApG) cross-links. Although the DNA recognition properties of members of the HMG-box family of proteins have been characterized with respect to their sequence specificity, the present work demonstrates that proteins with this domain can recognize particular DNA structures as well. The Pt-DNA SSRP described here is the human homolog of a recently identified mouse protein that binds to recombination signal sequences [Shirakata, M., Hüppi, K., Usuda, S., Okazaki, K., Yoshida, K. & Sakano, H. (1991) Mol. Cell. Biol. 11, 4528-4536]. These sequences have been postulated to form stem-loop structures, further implicating local bends and unwinding in DNA as a recognition target for HMG-box proteins. Expression analysis in a variety of tissues and cisplatin-resistant cell lines and the inability of cisplatin to induce the message in HeLa cells argue against a direct link between SSRP1 mRNA levels and the response of cells to the drug.
Isolation, structure, and activity of -Phe-Met-Arg-Phe-NH2 neuropeptides (designated calliFMRFamides) from the blowfly Calliphora vomitoria.
Authors: Duve H , Johnsen A H , Sewell J C , Scott A G , Orchard I , Rehfeld J F , Thorpe A
Published: May 2006

Thirteen neuropeptides varying in length from 7 to 11 residues and ending C-terminally in -Phe-Met-Arg-Phe-NH2 (calliFMRFamides 1-13) and one dodecapeptide ending in -Met-Ile-Arg-Phe-NH2 (calliMIRFamide 1) have been isolated from thoracic ganglia of the blowfly Calliphora vomitoria. Different repeating patterns of amino acid sequences enable the peptides to be arranged into distinct groups. One such group of five nonapeptides has the sequence Xaa-Pro-Xaa-Gln-Asp-Phe-Met-Arg-Phe-NH2. Three peptides in this group, with the N-terminal tripeptide sequences Thr-Pro-Gln-, Thr-Pro-Ser-, and Ser-Pro-Ser-, are able to induce fluid secretion from the isolated salivary gland of Calliphora at a concentration of 0.1 to 1 nM. However, two other members of this group with the N-terminal tripeptide sequences Lys-Pro-Asn- and Ala-Pro-Gly-, the latter being the most abundant peptide isolated, were inactive in this assay, as were all the other peptides isolated. This indicates that the N terminus (in addition to the C terminus as previously found for FMRFamides of other organisms) is crucial for at least some biological activities.
The Na+/H+ antiporter cytoplasmic domain mediates growth factor signals and controls "H(+)-sensing".
Authors: Wakabayashi S , Fafournoux P , Sardet C , Pouysségur J
Published: May 2006

The amiloride-sensitive Na+/H+ exchanger (NHE1 human isoform) is activated in response to diverse mitogenic and oncogenic signals presumably through phosphorylation. To get insight into the activating mechanism, a set of deletion mutants within the C-terminal cytoplasmic domain of NHE1 has been generated. These mutant forms expressed in antiporter-deficient fibroblasts revealed that deletion of the complete cytoplasmic domain (i) preserves amiloride-sensitive Na+/H+ exchange and activation by intracellular H+, (ii) reduces the affinity of the internal "H(+)-modifier site" in a manner mimicked by cellular ATP depletion, and (iii) abolishes growth factor-induced cytoplasmic alkalinization. We conclude that NHE1 can be separated into two distinct functional domains. One is an N-terminal transporter domain (T) that has all the features required to catalyze amiloride-sensitive Na+/H+ exchange with a built-in H(+)-modifier site. The other is a C-terminal cytoplasmic regulatory domain (R) that (i) determines the set point value of the exchanger and (ii) mediates growth factor signals by interacting with the "H(+)-sensor" in a phosphorylation-dependent manner.
Evidence for regulatory genes on mouse chromosome 7 that affect the quantitative expression of proteins in the fetal and newborn liver.
Authors: Giometti C S , Gemmell M A , Taylor J , Tollaksen S L , Angeletti R , Gluecksohn-Waelsch S
Published: May 2006

A series of deletions around the albino locus on mouse chromosome 7 is believed to include one or more regulatory genes that control the activities of a cluster of liver enzymes. To further characterize the functions of this region of the mouse genome, we have used quantitative two-dimensional electrophoresis to analyze the effects of two of these deletions, c3H and c14CoS, on the expression of liver proteins. More than 400 distinct protein gene products were quantitated in livers from fetal and newborn wild-type homozygous (cch/cch), heterozygous (cch/c3H or cch/c14CoS), and deletion homozygous (c3H/c3H or c14CoS/c14CoS) mice. Livers of fetal and newborn c3H heterozygotes and homozygous wild-type littermates produced qualitatively identical protein patterns after two-dimensional electrophoresis. In livers of c3H homozygous fetuses, however, abnormal amounts (either increased or decreased relative to homozygous wild-type and heterozygous littermates) of 29 proteins were found. Twenty-eight of these 29 protein anomalies were also found in livers of newborn c3H homozygotes. Livers of fetal and newborn mice homozygous for the c14CoS deletion, which overlaps the c3H deletion and produces a similar phenotype, expressed normal amounts of these proteins. One of the 29 proteins (MSN807) has an amino-terminal sequence similar to a 23-kDa translationally controlled protein abundant in mouse erythroleukemia and sarcoma-180 cells. These results suggest that normal chromosome 7 contains genes, located within the region of the c3H but not the c14CoS deletion, that regulate the abundance of specific proteins in the liver. These proteins cannot be related to the phenotypic alterations shared by the c3H and c14CoS deletions.
Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor alpha clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia.
Authors: Miller W H , Kakizuka A , Frankel S R , Warrell R P , DeBlasio A , Levine K , Evans R M , Dmitrovsky E
Published: May 2006

The characteristic t(15;17) of acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR-alpha) gene on chromosome 17 to a gene on chromosome 15 called PML, a putative transcription factor. This distinct translocation results in a fusion mRNA detected by Northern analysis. Two cDNAs have been isolated that differ in the extent of 3' PML nucleic acid sequence contained. This study describes a reverse transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha fusion transcript, which amplifies PML/RAR-alpha mRNA from APL cells with either reported breakpoint. DNA sequencing of the predominant RT-PCR products from 6 patients showed identical RAR-alpha exonic breakpoints and two PML breakpoints. This RT-PCR assay was positive in leukemic cells from 30/30 APL patients with the molecular rearrangement confirmed by cytogenetics or Northern analysis. In leukemic cells of patients with a morphologic diagnosis of APL lacking the t(15;17) by routine cytogenetics, a positive RT-PCR assay predicted clinical response to all-trans-retinoic acid (RA) therapy. Dilutional studies with leukemic cells that express (NB4) or do not express (HL-60) a PML/RAR-alpha fusion mRNA reveal that this RT-PCR assay detects the transcript from as little as 50 pg of total RNA. In APL cells from 5/6 patients treated with RA alone, a complete response by clinical and cytogenetic criteria accompanied a persistently positive RT-PCR assay. This preceded relapse by 1-6 months. RT-PCR for PML/RAR-alpha mRNA provides a more-sensitive test for the t(15;17) than routine cytogenetics or Northern analysis. This molecular rearrangement detected by RT-PCR best defines this RA-responsive malignancy. The RT-PCR assay for the PML/RAR-alpha transcript yields important diagnostic and prognostic information in the management of APL patients.
p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15.
Authors: Parker L L , Atherton-Fessler S , Piwnica-Worms H
Published: May 2006

p107wee1 is a protein kinase that functions as a dose-dependent inhibitor of mitosis through its interactions with p34cdc2 in Schizosaccharomyces pombe. To characterize the kinase activity of p107wee1, its carboxyl-terminal catalytic domain was purified to homogeneity from overproducing insect cells. The apparent molecular mass of the purified protein (p37wee1KD) was determined to be approximately 37 kDa by gel filtration, consistent with it being a monomer. Serine and tyrosine kinase activities cofiltered with p37wee1KD, demonstrating that p107wee1 is a dual-specificity kinase. In vitro, p107wee1 phosphorylated p34cdc2 on Tyr-15 only when p34cdc2 was complexed with cyclin. Neither monomeric p34cdc2 nor a peptide containing Tyr-15 was able to substitute for the p34cdc2/cyclin complex in this assay. Furthermore, the phosphorylation of p34cdc2 by p107wee1 in vitro inhibited the histone H1 kinase activity of p34cdc2. These results indicate that p107wee1 functions as a mitotic inhibitor by directly phosphorylating p34cdc2 on Tyr-15 and that the preferred substrate for phosphorylation is the p34cdc2/cyclin complex.