We have characterized three distinct proteins present in HeLa cell extracts that specifically recognize different subsets of transcriptional elements containing the pentanucleotide sequence CCAAT. One of these CCAAT-binding proteins, CP1, binds with high affinity to CCAAT elements present in the human alpha-globin promoter and the adenovirus major late promoter (MLP). A second protein, CP2, binds with high affinity to a CCAAT element present in the rat gamma-fibrinogen promoter. Finally, the third CCAAT-binding protein is nuclear factor I (NF-I), a cellular DNA-binding protein that binds to the adenovirus origin of replication and is required for the initiation of adenoviral replication. CP1, CP2, and NF-I are distinct activities in that each binds to its own recognition site with an affinity that is at least three orders of magnitude higher than that with which it binds to the recognition sites of the other two proteins. Surprisingly, CP1, CP2, and NF-I each appear to recognize their binding site with highest affinity as a multisubunit complex composed of heterologous subunits. In the case of CP1, two different types of subunits form a stable complex in the absence of a DNA-binding site. Moreover, both subunits are present in the CP1-DNA complex. We thus propose the existence of a family of related multisubunit CCAAT-binding proteins that are composed of heterologous subunits.

The AICD (amyloid precursor protein [APP] intracellular domain) and C31, the caspase-cleaved C-terminal fragment of APP, have been found in the brains of patients with Alzheimer's disease (AD). Here, we demonstrate for the first time that the C-terminal fragments of APP (AICD [C57, C59] and C31) exert neurotoxicity on differentiated PC 12 cells and rat primary cortical neurons by inducing the expression of glycogen synthase kinase 3beta, forming a ternary complex with Fe65 and CP2/LSF/LBP1 in the nucleus, whereas deletion mutants and a point mutant with Y682G of the YENPTY domain, a Fe65 binding domain, do not. Moreover, expression of APP770 and Swedish mutant form of APP increased the levels of C-terminal fragments of APP (APP-CTs) in neuronal cells and also induced the up-regulation of glycogen synthase kinase-3beta at both the mRNA and the protein levels. In addition, we show that CP2/LSF/LBP1 binding site (nt +0 to approximately +10) in human glycogen synthase kinase 3beta promoter region is essential for the induction of the gene transcription by APP-CTs. The neurotoxicities induced by APP-CTs (AICD and C31) were accompanied by an increase in the active form of glycogen synthase kinase-3beta, and by the induction of tau phosphorylation and a reduction in nuclear beta-catenin levels, and led to apoptosis.

Changes in [Mg2+] in a millimolar range have a significant inverse effect on the Ca2+- (or Sr2+)activated tension generation of skeletal muscle fibers. Single frog (Rana pipiens) semitendinosus muscle fibers were "skinned" (sarcolemma removed) and contracted isometrically in bathing solutions of varying [Ca2+] or [Sr2+] and [Mg2+] but a constant pH, [MgATP2-], [K+], [CP2-], [CPK], and ionic strength. Ca2+- (or Sr2+-)activated steady-state tensions were recorded for three [Mg2+]'s: 5 X 10(-5)M, 1 X 10(-3) M, and 2 X 10(-3) M; and these tensions were expressed as the percentages of maximum tension generation of the fibers for the same [Mg2+]. Maximum tension was not affected by [Mg2+] within Ca2+-activating or Sr2+-activating sets of solutions; however, the submaximum Ca2+-(or Sr2+)activated tension is strongly affected in an inverse fashion by increasing [Mg2+]. Mg2+ behaves as a competitive inhibitor of Ca2+ and also affects the degree of cooperativity in the system. At [Mg2+] = 5 X 10(-5)M the shape of tension versus [Ca2+] (or [Sr2+]) curve showed evidence of cooperativity of Ca2+ (or Sr2+) binding or activation of the contractile system. As [Mg2+] increased, the apparent affinity for Ca2+ or Sr2+ and cooperativity of the contractile system declined. The effect on cooperativity suggests that as [Mg2+] decreases a threshold for Ca2+ activation appears.

Mouse embryonic stem cell (mESC) self-renewal can be maintained by activation of the leukaemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (Stat3) signalling pathway or dual inhibition (2i) of glycogen synthase kinase 3 (Gsk3) and mitogen-activated protein kinase kinase (MEK). Several downstream targets of the pathways involved have been identified that when individually overexpressed can partially support self-renewal. However, none of these targets is shared among the involved pathways. Here, we show that the CP2 family transcription factor Tfcp2l1 is a common target in LIF/Stat3- and 2i-mediated self-renewal, and forced expression of Tfcp2l1 can recapitulate the self-renewal-promoting effect of LIF or either of the 2i components. In addition, Tfcp2l1 can reprogram post-implantation epiblast stem cells to naïve pluripotent ESCs. Tfcp2l1 upregulates Nanog expression and promotes self-renewal in a Nanog-dependent manner. We conclude that Tfcp2l1 is at the intersection of LIF- and 2i-mediated self-renewal pathways and plays a critical role in maintaining ESC identity. Our study provides an expanded understanding of the current model of ground-state pluripotency.

Two extended haplotypes exist across the tau gene-H1 and H2-with H1 consistently associated with increased risk of progressive supranuclear palsy (PSP). Using 15 haplotype tagging SNPs (htSNPs), capturing >95% of MAPT haplotype diversity, we performed association analysis in a US sample of 274 predominantly pathologically confirmed PSP patients and 424 matched control individuals. We found that PSP risk is associated with one of two major ancestral H1 haplotypes, H1B, increasing from 14% in control individuals to 22% in PSP patients (P<0.001). In young PSP patients, the H1B risk could be localized to a 22 kb regulatory region in intron 0 (P<0.001) and could be fully explained by one SNP, htSNP167, creating a LBP-1c/LSF/CP2 site, shown to regulate the expression of genes in other neurodegenerative disorders. Luciferase reporter data indicated that the 182 bp conserved regulatory region, in which htSNP167 is located, is transcriptionally active with both alleles differentially influencing expression. Further, we replicated the htSNP167 association in a second, independently ascertained US PSP patient-control sample. However, the htSNP association showed that H1 risk alone could not explain the overall differences in H1 and H2 frequencies in PSP patients and control individuals. Thus, risk variants on different H1 htSNP haplotypes and protective variants on H2 contribute to population risk for PSP.

The FE65s (FE65, FE65L1, and FE65L2) are a family of multidomain adaptor proteins that form multiprotein complexes with a range of functions. FE65 is brain-enriched, whereas FE65L1 and FE65L2 are more widely expressed. All three members contain a WW domain and two PTB domains. Through the PTB2 domain, they all interact with the Alzheimer's disease amyloid precursor protein (APP) intracellular domain (AICD) and can alter APP processing. After sequential proteolytic processing of membrane-bound APP and release of AICD to the cytoplasm, FE65 can translocate to the nucleus to participate in gene transcription events. This role is further mediated by interactions of FE65 PTB1 with the transcription factors CP2/LSF/LBP1 and Tip60 and the WW domain with the nucleosome assembly factor SET. However, FE65 target genes have not yet been confirmed. The FE65 PTB1 domain also interacts with two cell surface lipoproteins receptors, the low-density lipoprotein receptor-related protein (LRP) and ApoEr2, forming trimeric complexes with APP. The FE55 WW domain also binds to mena, through which it functions in regulation of the actin cytoskeleton, cell motility, and neuronal growth cone formation. While single knockout mice appear normal, double FE65(-/-)/FE65L1(-/-) mice have substantial neurodevelopmental defects. These include heterotopic neurons and axonal pathfinding defects, findings similar to findings in both Mena and triple APP:APLP1:APLP2 knockout mice and also lissencephalopathies in humans. Thus APPs, FE65s, and mena may act together in a developmental signalling pathway. This article reviews the known functions of the FE65 family and their role in APP function and Alzheimer's disease.

The androgen receptor (AR) mediates androgen action and plays a central role in the proliferation of specific cancer cells. We demonstrated recently that AR mRNA stability is a major determinant of AR gene expression in prostate and breast cancer cells and that androgens differentially regulate AR mRNA decay dependent on cell type (Yeap, B. B., Kreuger, R. G., Leedman, P. J. (1999) Endocrinology 140, 3282-3291). Here, we have identified a highly conserved UC-rich region in the 3-untranslated region of AR mRNA that contains a 5'-C(U)(n)C motif and a 3'-CCCUCCC poly(C)-binding protein motif. In transfection studies with LNCaP human prostate cancer cells, the AR UC-rich region reduced expression of a luciferase reporter gene. The AR UC-rich region was a target for cytoplasmic and nuclear RNA-binding proteins from human prostate and breast cancer cells as well as human testicular and breast cancer tissue. One of these proteins is HuR, a ubiquitously expressed member of the Elav/Hu family of RNA-binding proteins involved in the stabilization of several mRNAs. Poly(C)-binding protein-1 and -2 (CP1 and CP2), previously implicated in the control of mRNA turnover and translation, also bound avidly to the UC-rich region. Mutational analysis of the UC-rich region identified specific binding motifs for both HuR and the CPs. HuR and CP1 bound simultaneously to the UC-rich RNA and in a cooperative manner. Immunoprecipitation studies confirmed that each of these proteins associated with AR mRNA in prostate cancer cells. In summary, we have identified and characterized a novel complex of AR mRNA-binding proteins that target the highly conserved UC-rich region. The binding of HuR, CP1, and CP2 to AR mRNA suggests a role for each of these proteins in the post-transcriptional regulation of AR expression in cancer cells.

The Drosophila transcription factor Grainyhead regulates several key developmental processes. Three mammalian genes, CP2, LBP-1a and LBP-9 have been previously identified as homologues of grainyhead. We now report the cloning of two new mammalian genes (Mammalian grainyhead (MGR) and Brother-of-MGR (BOM)) and one new Drosophila gene (dCP2) that rewrite the phylogeny of this family. We demonstrate that MGR and BOM are more closely related to grh, whereas CP2, LBP-1a and LBP-9 are descendants of the dCP2 gene. MGR shares the greatest sequence homology with grh, is expressed in tissue-restricted patterns more comparable to grh and binds to and transactivates the promoter of the human Engrailed-1 gene, the mammalian homologue of the key grainyhead target gene, engrailed. This sequence and functional conservation indicates that the new mammalian members of this family play important developmental roles.

BACKGROUND

The objectives of this study were to compare both subjective clinical outcomes and the objective stress response of laparoscopic and open abdominal rectopexy in patients with full-thickness rectal prolapse. Abdominal rectopexy for patients with rectal prolapse is well suited for a laparoscopic approach as no resection or anastomosis is necessary.

METHODS

Forty patients with a full-thickness rectal prolapse were randomized before operation to a laparoscopic group and an open group. They agreed to conform to a clinical pathway (CP) of liquid diet (CP1) and full mobility (CP2) on day 1, solid diet (CP3) on day 2 and discharge (CP4) before day 5. Their compliance was monitored by an assessor blinded to the operative group, who also rated pain and mobility. Patient-controlled morphine use was documented. Neuroendocrine and immune stress response and respiratory function were measured.

RESULTS

Some 75 per cent of all clinical pathway objectives of early recovery were achieved in the laparoscopic group compared with 37 per cent in the open group (P < 0.01). Significant differences in favour of laparoscopy were noted with regard to narcotic requirements, and pain and mobility scores. Differences in objective measures of stress response favouring laparoscopy were found for urinary catecholamines, interleukin 6, serum cortisol and C-reactive protein. No differences were noted in respiratory function but significant respiratory morbidity was greater in the open group (P < 0.05). None of the measured outcomes, subjective or objective, favoured the open group apart from operating time, which was significantly shorter (153 versus 102 min; P < 0.01).

CONCLUSIONS

This study has demonstrated significant subjective and objective differences in favour of a laparoscopic technique for abdominal rectopexy. The advantages were all short term but no evidence of any adverse effect on longer-term outcomes was observed.

Strain AK127 is a developmental mutant of Dictyostelium discoideum that was isolated by restriction enzyme-mediated integration (REMI). Mutant cells aggregate normally but are unable to proceed past the loose aggregate stage. The cloned gene, lagC (loose aggregate C), encodes a novel protein of 98 kD that contains an amino-terminal signal sequence and a putative carboxy-terminal transmembrane domain. The mutant strain AK127 shows no detectable lagC transcript upon Northern analysis, indicating that the observed phenotype is that of a null allele. Expression of the lagC cDNA in AK127 cells complements the arrest at the loose aggregate stage, indicating that the mutant phenotype results from disruption of the lagC gene. In wild-type cells, lagC mRNA is induced at the loose aggregate stage and is expressed through the remainder of development. lagC- null cells aggregate but then disaggregate and reaggregate to form small granular mounds. Mature spores are produced at an extremely low efficiency (< 0.1% of wild type), appearing only after approximately 72 hr, whereas wild-type strains produce mature spores by 26 hr. lagC- null cells accumulate reduced levels of transcripts for the prestalk-enriched genes rasD and CP2 and do not express the DIF-induced prestalk-specific gene ecmA or the cAMP-induced prespore-specific gene SP60 to significant levels. In chimeric organisms resulting from the coaggregation of lagC- null and wild-type cells, cell-type-specific gene expression is rescued in the lagC- null cells; however, lagC- prespore cells are localized to the posterior of the prespore region and do not form mature spores, suggesting that LagC protein has both no cell-autonomous and cell-autonomous functions. Overexpression of lagC from an actin promoter in both wild-type and lagC- cells causes a delay at the tight aggregate stage, the first stage requiring LagC activity. These results suggest that the LagC protein functions as a nondiffusible cell-cell signaling molecule that is required for multicellular development.

Antibodies prepared against peptides CP2, CP4, and CP5, which occur within the first 1522 amino acid residues of the alpha 1 subunit of dihydropyridine-sensitive skeletal muscle calcium channels, specifically recognized a 175-kDa form of the alpha 1 subunit in immunoblots and immunoprecipitation experiments. In contrast, antibodies prepared against peptide CP1, which represents the C-terminal 18 amino acid residues predicted by cloning and sequence analysis of the alpha 1 subunit, recognized a minor, previously undescribed 212-kDa protein, which is the size predicted for the full length of the alpha 1 subunit from cDNA cloning [Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1987) Nature (London) 328, 313-318]. Both the 175-kDa and 212-kDa forms were phosphorylated by cAMP-dependent protein kinase and both were present in isolated transverse tubule membranes. The 175-kDa form may arise from posttranslational proteolytic cleavage of the C terminus of the 212-kDa form of the alpha 1 subunit predicted by cDNA cloning and sequence analysis. Partial amino acid sequencing of the 54-kDa beta subunit of the calcium channel indicated this protein was not derived from the proteolytically cleaved C terminus of the alpha 1 subunit. This analysis identified a threonine residue in the sequence (Lys/Arg)-Arg-Pro-Thr-Pro of the beta subunit that was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of this residue in the beta subunit may play a role in modulation of calcium channel function. Separate functional roles of the 175-kDa form of the alpha 1 subunit in excitation-contraction coupling and of the 212-kDa form in ion conductance are proposed.

Amyloid precursor protein (APP) has eight potential phosphorylation sites in its cytoplasmic domain. Recently, it has demonstrated that the constitutive phosphorylation of APP at T668 (APP695 isoform numbering) was observed specifically in the brain. Neuron-specific phosphorylation of APP at T668 is thought to be important for neuronal functions of APP, although its exact physiological significance remains to be clarified. In this study, we show that the phosphorylation of the APP intracellular domain (AICD) at T668 is essential for its binding to Fe65 and its nuclear translocation and affects the resultant neurotoxicity, possibly mediated through the induction of glycogen synthase kinase 3beta and tau phosphorylation by enhancing the formation of a ternary complex with Fe65 and CP2 transcription factor. Taken together, these results suggest that the phosphorylation of AICD at T668 contributes to the neuronal degeneration in Alzheimer's disease (AD) by regulating its translocation into the nucleus and then affects neurodegeneration; therefore, the specific inhibitor of T668 phosphorylation might be the target of AD therapy.

LBP-1 is a cellular protein which binds strongly to sequences around the human immunodeficiency virus type 1 (HIV-1) initiation site and weakly over the TATA box. We have previously shown that LBP-1 represses HIV-1 transcription by inhibiting the binding of TFIID to the TATA box. Four similar but distinct cDNAs encoding LBP-1 (LBP-1a, -b, -c, and -d) have been isolated. These are products of two related genes, and each gene encodes two alternatively spliced products. Comparison of the amino acid sequence of LBP-1 with entries in the available protein data bases revealed the identity of LBP-1c to alpha-CP2, an alpha-globin transcription factor. These proteins are also homologous to Drosophila melanogaster Elf-1/NTF-1, an essential transcriptional activator that functions during Drosophila embryogenesis. Three of the recombinant LBP-1 isoforms show DNA binding specificity identical to that of native LBP-1 and bind DNA as a multimer. In addition, antisera raised against recombinant LBP-1 recognize native LBP-1 from HeLa nuclear extract. Functional analyses in a cell-free transcription system demonstrate that recombinant LBP-1 specifically represses transcription from a wild-type HIV-1 template but not from an LBP-1 mutant template. Moreover, LBP-1 can function as an activator both in vivo and in vitro, depending on the promoter context. Interestingly, one isoform of LBP-1 which is missing the region of the Elf-1/NTF-1 homology is unable to bind DNA itself and, presumably through heteromer formation, inhibits binding of the other forms of LBP-1, suggesting that it may function as a dominant negative regulator.

CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1.

The neural protein Fe65 possesses three putative protein-protein interaction domains: one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1 and PID2); the most C-terminal of these domains (PID2) interacts in vivo with the Alzheimer's beta-amyloid precursor protein, whereas the WW domain binds to Mena, the mammalian homolog of Drosophila-enabled protein. By the interaction trap procedure, we isolated a cDNA clone encoding a possible ligand of the N-terminal PID/PTB domain of Fe65 (PID1). Sequence analysis of this clone revealed that this ligand corresponded to the previously identified transcription factor CP2/LSF/LBP1. Co-immunoprecipitation experiments demonstrated that the interaction between Fe65 and CP2/LSF/LBP1 also takes place in vivo between the native molecules. The localization of both proteins was studied using fractionated cellular extracts. These experiments demonstrated that the various isoforms of CP2/LSF/LBP1 are differently distributed among subcellular fractions. At least one isoform, derived from alternative splicing (LSF-ID), is present outside the nucleus; Fe65 was found in both fractions. Furthermore, transfection experiments with an HA-tagged CP2/LSF/LBP1 cDNA demonstrated that Fe65 interacts also with the nuclear form of CP2/LSF/LBP1. Considering that the analysis of Fe65 distribution in fractionated cell extracts demonstrated that this protein is present both in nuclear and non-nuclear fractions, we examined the expression of Fe65 deletion mutants in the two fractions. This analysis allowed us to observe that a small region N-terminal to the WW domain is phosphorylated and is necessary for the presence of Fe65 in the nuclear fraction.

The thymidylate synthase (TS) gene, which is induced at the G(1)-S transition in growth-stimulated cells, encodes an enzyme that is essential for DNA replication and cell survival. Here we demonstrate that LSF (LBP-1c, CP2) binds to sites within the TS promoter and intronic regions that are required for this induction. Mutation of the LSF binding sites inhibits G(1)-S induction of mRNA derived from a TS minigene. Furthermore, expression of dominant-negative LSF (LSFdn) prevents the increase in TS enzyme levels during G(1)-S, and induces apoptosis in growth- stimulated mouse and human cell lines. Such apoptosis can be prevented either by circumventing the TS requirement through addition of low concentrations of thymidine, or by coexpression of the TS gene driven by a heterologous promoter. Induction of apoptosis by LSFdn parallels the process known as thymineless death, which is induced by the TS inhibitor and chemotherapeutic drug 5-fluorodeoxyuridine. Thus, LSF is a novel regulatory factor that supports progression through S-phase by targeting a single gene that is critical for cell survival.

The human stage selector protein (SSP) has been implicated in the developmental regulation of the globin genes. Binding of SSP to the stage selector element (SSE) in the proximal gamma-globin promoter is integral to the competitive silencing of a linked beta-promoter in embryonic/fetal stage erythroleukemia (K562) cells. We now report the biochemical purification of SSP from K562 cell nuclear extract and demonstrate that the ubiquitously expressed transcription factor CP2 is pivotal to, but not sufficient for, SSP binding activity. Although addition of anti-CP2 antiserum disrupts the formation of the SSP-SSE complex in the electrophoretic mobility shift assay (EMSA), recombinant CP2 fails to bind to the SSE. Binding of CP2 to the SSE requires a heterodimeric partner present in K562 cells. We have defined the molecular weight of the partner protein as 40-45 kDa in UV and protein cross-linking experiments. An element analogous to the human SSE has previously been demonstrated in the chicken beta A-gene-promoter. The effects of this element are dependent on the binding of the chicken stage selector protein, NF-E4. Comparative studies between human CP2 and chicken NF-E4 demonstrate homology between the protein complexes. SSP binds to the chicken SSE and formation of this complex is ablated by the addition of anti-CP2 antiserum or a monoclonal antibody to NF-E4. Western analysis of partially purified NF-E4 using anti-CP2 antiserum or the NF-E4 monoclonal antibody both demonstrate a dominant band at 66 kDa. Similarly, the NF-E4 antibody recognizes the 66 kDa human CP2 protein in Western analysis of the SSP-SSE complex.(ABSTRACT TRUNCATED AT 250 WORDS)

The stage selector protein (SSP) is a heteromeric complex involved in preferential expression of the human gamma-globin genes in fetal-erythroid cells. We have previously identified the ubiquitous transcription factor CP2 as a component of this complex. Using the protein dimerization domain of CP2 in a yeast two-hybrid screen, we have cloned a novel gene, NF-E4, encoding the tissue-restricted component of the SSP. NF-E4 and CP2 coimmunoprecipitate from extract derived from a fetal-erythroid cell line, and antiserum to NF-E4 ablates binding of the SSP to the gamma promoter. NF-E4 is expressed in fetal liver, cord blood, and bone marrow and in the K562 and HEL cell lines, which constitutively express the fetal globin genes. Enforced expression of NF-E4 in K562 cells and primary erythroid progenitors induces endogenous fetal globin gene expression, suggesting a possible strategy for therapeutic intervention in the hemoglobinopathies.

The neurotoxicity of amyloid-beta protein (Abeta) is widely regarded as one of the fundamental causes of neurodegeneration in Alzheimer's disease (AD). This toxicity is related to Abeta aggregation into oligomers, protofibrils and fibrils. Recent studies suggest that intracellular Abeta, which causes profound toxicity, could be one of the primary therapeutic targets in AD. So far, no compounds targeting intracellular Abeta have been identified. We have investigated the toxicity induced by intracellular Abeta in a neuroblastoma MC65 line and found that it was closely related to intracellular accumulation of oligomeric complexes of Abeta (Abeta-OCs). We further identified a cell-permeable tricyclic pyrone named CP2 that ameliorates this toxicity and significantly reduces the levels of Abeta-OCs. In aqueous solution, CP2 attenuates Abeta oligomerization and prevents the oligomer-induced death of primary cortical neurons. CP2 analogs represent a new class of promising compounds for the amelioration of Abeta toxicities within both intracellular and extracellular sites.

The Drosophila gene grainyhead is the founding member of a large family of genes encoding developmental transcription factors that are highly conserved from fly to human. The family consists of two main branches, with grainyhead as the ancestral gene for one branch and the recently cloned Drosophila CP2 as the ancestral gene for the other. We now extend this family with the identification of another novel mammalian member, Sister-of-Mammalian Grainyhead (SOM), which is phylogenetically aligned with grainyhead. SOM is closely related to the other mammalian homologues of grainyhead, including Mammalian Grainyhead (MGR) and Brother-of-MGR, sharing a high degree of sequence identity with these factors in the functional DNA-binding, protein dimerization and activation domains. Protein interaction studies demonstrate that SOM can heterodimerize with MGR and Brother-of-MGR, but not with the more distant members of the family. Like grainyhead, the SOM gene too produces several distinct isoforms with differing functional properties through alternative splicing. The tissue distributions of these isoforms differ and all display highly restricted expression patterns. These findings indicate that SOM, like its family members, may play important roles in mammalian development.

This study reports outcomes of allogeneic hematopoietic stem cell transplantation with reduced-intensity conditioning (RIC) in 186 patients with chronic myeloid leukemia (CML) from the European Group for Blood and Marrow Transplantation (EBMT). The median age was 50 years, and 64% were in first chronic phase (CP1), CP2 13%, accelerated phase 17%, and blast crises 6%. The median EBMT transplant score was 3. The day 100 transplantation-related mortality (TRM) was 6.1% (confidence interval [CI], 3.4%-11%) but rose to 23.3% (CI, 14%-27%) at 2 years. Fludarabine, busulfan, and antithymocyte globulin (Fd/Bu/ATG) was associated with the lowest TRM of 11.6% (CI, 4.7%-11%) at 1 year. Acute graft-versus-host disease (GvHD) grade II to IV occurred in 32% and chronic GvHD in 43% (extensive in 24%). ATG was associated with a lower incidence of chronic GvHD (cGvHD). The overall survival (OS) and progression-free survival (PFS) at 3 years were 58% (CI, 50%-66%) and 37% (CI, 30%-45%), respectively. Adverse OS was associated with advanced disease (relative risk [RR], 3.4). PFS was inferior in advanced disease (RR, 2.7) and a trend to improved outcomes with Fd/Bu/ATG (RR, 0.58). RIC allografts are feasible in CML in first or second CP. Since no other RIC regimen demonstrated superiority, Fd/Bu/ATG should be considered as baseline in future prospective trials.

In this study we addressed the question of the intracellular localization of Fe65, an adaptor protein interacting with the beta-amyloid precursor protein (APP) and with the transcription factor CP2/LSF/LBP1. By using tagged Fe65 expression vectors, we observed that a significant fraction of Fe65 is localized in the nucleus of transfected COS7 cells. Furthermore, the isolation of nuclei from untransfected PC12 cells allowed us to observe that a part of the endogenous Fe65 is present in the nuclear extract. The analysis of Fe65 mutant constructs demonstrated that the region of the protein required for its nuclear translocation includes the WW domain, and that, on the other hand, a small fragment of 100 residues, including this WW domain, contains enough structural information to target a reporter protein (green fluorescent protein (GFP)-GFP) to the nucleus. To evaluate whether the Fe65-APP interaction could affect Fe65 intracellular trafficking, COS7 cells were cotransfected with APP(695) or APP(751) and with GFP-Fe65 expression vectors. These experiments demonstrated that Fe65 is no longer translocated to the nucleus when the cells overexpress APP, whereas the nuclear targeting of GFP-Fe65 mutants, unable to interact with APP, is unaffected by the coexpression of APP, thus suggesting that the interaction with APP anchors Fe65 in the cytosol.

Two murine mAb were prepared against human mast cell carboxypeptidase (HMC-CP) purified from human skin, and were termed CP1 and CP2, respectively. Double immunohistochemical labeling of Carnoy's-fixed sections of human skin, lung, and gastrointestinal tissue with CP1 and CP2, respectively, and with a murine monoclonal antitryptase antibody demonstrated that HMC-CP was selectively present in a subset of human mast cells. Double labeling experiments with CP1 and CP2, respectively, and a murine anti-chymase mAb demonstrated the presence of HMC-CP in the tryptase-positive, chymase-positive mast cell type (MCTC) only. Immunohistochemical labeling of peripheral blood leukocytes resulted in staining of monocytes with CP2 but not with CP1. In addition to chymase and a cathepsin-G like proteinase, HMC-CP is another neutral protease that is selectively present in the MCTC tryptase-positive, chymase-positive mast cells type of mast cell, thus extending the biochemical definition of human mast cell heterogeneity.

The nucleotide sequences of cDNAs coding for human salivary proline-rich proteins (PRPs) were determined. Clones cP1 and cP2 contain repetitive regions in which sites for the restriction enzyme HaeIII occur repeatedly; they code for the precursors of acidic PRPs. Clones cP3 to cP7 contain repetitive regions in which BstNI sites occur repeatedly; they code for precursors of basic and glycosylated PRPs. The clones cP3, cP4, and cP5 are identical except that cP4 and cP5 are missing 399 and 459 base pairs, respectively, from the repetitive region of cP3. The sequences at these deletion end points are homologous to the consensus sequences of RNA splicing donor and acceptor sites. This strongly suggests that all three cDNAs are derived from the transcript of a single gene via differential RNA splicing. All of the precursor proteins share a feature--the N-terminal region, following the signal peptide, is acidic, while the remainder of the molecule, made of proline-rich repeats of about 21 amino acids, is basic. Each precursor can generate multiple PRPs by various post-translational cleavages on the carboxylic side of specific arginine residues. The data show how differential RNA splicing and post-translational cleavages could generate a large number of proteins, such as those found in saliva, from a much smaller number of genes.