Human ERG-2 protein is a phosphorylated DNA-binding protein--a distinct member of the ets family

We describe the identification of the ERG-2 gene products using an antibody raised against recombinant human ERG-2 protein. ERG-2 is a nuclear phosphoprotein and binds to purine-rich sequences (C/G)(C/a)GG-AA(G/a)T. ERG-2 protein, with a half-life of 21 h, is considerably more stable than the short-lived ETS-1 or ETS-2 proteins. Its phosphorylation is stimulated by phorbol myristate acetate (PMA), but not by Ca2+ ionophore treatment. ETS-1 protein is phosphorylated by Ca(2+)-dependent events, whereas ERG-2 protein is phosphorylated by activation of protein kinase C, suggesting their involvement in distinct signal transduction mechanisms. The expression of ERG-2 protein is restricted to few cell types and is high in early myeloid cells, indicating that it may function at an early stage of hematopoietic lineage determination. The DNA-binding sequence for ERG-2 protein is identified by using a random oligonucleotide selection procedure. The selected sequence is very similar to the binding sequence determined for human ETS-1 using the same method. Like other ets proteins, ERG-2 is a sequence-specific DNA-binding protein and is expressed at higher levels in early myeloid cells than in mature lymphoid cells. These results suggest that it may act as a regulator of genes required for maintenance and/or differentiation of early hematopoietic cells.     

A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export

Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin beta superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.     

The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family

The ability of the shoot apical meristem to multiply and distribute its meristematic potential through the formation of axillary meristems is essential for the diversity of forms and growth habits of higher plants. In the lateral suppressor mutant of tomato the initiation of axillary meristems is prevented, thus offering the unique opportunity to study the molecular mechanisms underlying this important function of the shoot apical meristem. We report here the isolation of the Lateral suppressor gene by positional cloning and show that the mutant phenotype is caused by a complete loss of function of a new member of the VHIID family of plant regulatory proteins.     

CD63, a member of tetraspan transmembrane protein family, induces cellular spreading by reaction with monoclonal antibody on substrata

We cloned mouse LOX-1 cDNA to take advantage of a gene-targeting technique to clarify the role of LOX-1 in vivo. Mouse LOX-1 was composed of 363 amino acids and had a C-type lectin domain type II membrane protein structure. Mouse LOX-1 had triple repeats of the sequence in the extracellular "Neck domain," which is unlike human and bovine LOX-1. LOX-1 bound oxidized LDL with two classes of binding affinity in the presence of serum. The binding component with the higher affinity showed the lowest value of Kd among the known receptors for oxidized LDL. In the absence of serum, the high affinity component disappeared, suggesting that an unknown co-factor in serum is essential for efficient uptake of oxidized LDL by endothelial cells. A low concentration of unlabeled oxidized LDL displaced 125I-labeled oxidized LDL more efficiently in the presence of serum than in the absence of serum. The co-factor in the serum may be involved in the pathophysiology of atherosclerosis in addition to the oxidation of LDL.     

A fibroadenoma with a t(4;12) (q27;q15) affecting the HMGI-C gene, a member of the high mobility group protein gene family

The tumor-initiating activities of the methanol extracts of polyetherurethanes (PEUs) were first detected in the presence of 12-0-tetradecanyl-phorbol-13-acetate (TPA) using Balb 3T3 transformation assay. A model hard segment of PEUs, 4,4'-di(ethoxycarboamide) diphenylmethane (MDU), showed initiating activity, while chemical moieties other than the hard segment were shown to be negative in the test. The transformation assay was carried out using glass dishes half coated with two different PEUs, PU4 and PU8. In the presence of TPA, the transforming activities correlated with the tumorigenic potential in the rat implantation study on the coated surface of PEUs, but not on the uncoated glass area. From these results it was concluded that initiation was caused by the hard-segment moiety such as MDU structure derived not only from the leachable extracts but also from the biodegradable substances by the direct interaction of cells with the coated materials.     

The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family

A physiological role for paraoxonase (PON1) is still uncertain, but it catalyzes the hydrolysis of toxic organophosphates. Evidence that the human genome contains two PON1-like genes, designated PON2 and PON3, is presented here. Human PON1 and PON2 each have nine exons, and the exon/intron junctions occur at equivalent positions. PON1 and PON2 genes are both on chromosome 7 in human and on chromosome 6 in the mouse. Turkey and chicken, like most birds, lack paraoxonase activity and are very susceptible to organophosphates. However, they have a PON-like gene with approximately 70% identity with human PON1, PON2, and PON3. Another unexpected finding is that the deduced amino acid sequences of PON2 in human, mouse, dog, turkey, and chicken and of human PON3 are all missing the amino acid residue 105, which is lysine in human PON1. The expanded number of PON genes will have important implications for future experiments designed to discover the individual functions, catalytic properties, and physiological roles of the paraoxonases.     

Identification and characterization of a heat shock protein 70 family member in channel catfish (Ictalurus punctatus)

The primary objective of this study was to evaluate the electrophysiologic effects of large-dose propofol, used as the sole anesthetic in patients with epilepsy. Nine patients with medically intractable complex partial epilepsy undergoing a three-stage approach to the surgical management of epilepsy were recruited. State I involved placement of the intracranial electrode array, while Stage II consisted of extraoperative localization of the seizure focus. The patients were studied during induction of anesthesia for Stage III (removal of electrodes and resection of seizure focus). Unpremedicated patients were induced with a propofol infusion (0.5 mg.kg-1.min-1) until one of the following occurred: 1) electrical seizure activity, 2) burst suppression, or 3) total dose of 10 mg/mg. Electrocorticography (ECoG) was recorded continuously during this period. Two patients were excluded from the study after experiencing delayed awakening after the Stage I procedure. Both had received propofol along with other anesthetics. No ECoG evidence of seizure activity was detected in the seven patients completing the study. Burst suppression was attained in six patients using a mean dose of 5.7 mg/kg +/- 2.6. We conclude that large dose propofol alone does not trigger electrical epileptiform activity on the ECoG of seizure patients.     

Calcium-induced exposure of a hydrophobic surface of mouse ALG-2, which is a member of the penta-EF-hand protein family

ALG-2 is a 22 kDa EF-hand type Ca2+-binding protein associated with lymphocyte apoptosis. Comparison of the primary structure of ALG-2 with those of EF-hand type proteins revealed that it belongs to the penta-EF-hand (PEF) protein family including the small subunit of calpain. We established a convenient method for the purification of the recombinant mouse ALG-2 expressed in Escherichia coli. The recombinant protein was first pelleted from a lysate in the absence of a Ca2+-chelator, and then extracted with buffer containing EDTA/EGTA followed by purification by conventional column chromatographies. Estimation of the molecular mass by gel filtration suggested that the recombinant ALG-2 occurred as a monomeric form. Ca2+-dependent precipitation was blocked by inclusion of non-ionic detergent Triton X-100, suggesting hydrophobic self-aggregation at high concentrations of the protein. The N-terminal deletion mutant lacking the hydrophobic non-PEF region was found to be more soluble than the wild type in the presence of Ca2+. Analysis using a fluorescent hydrophobicity probe indicated that ALG-2 exposed a hydrophobic surface in a Ca2+-concentration dependent manner, the half-maximal effect occurring at approximately 6 microM. Mg2+ was not effective for the conformational change. On Western blotting, ALG-2 was detected in particulate fractions from cultured mammalian cells, suggesting the association of the protein with macromolecules in the cells.     

Control of G2/M transition in Xenopus by a member of the p21-activated kinase (PAK) family: a link between protein kinase A and PAK signaling pathways?

X-PAKs are involved in negative control of the process of oocyte maturation in Xenopus (). In the present study, we define more precisely the events targetted by the kinase in the inhibition of the G2/M transition. We show that microinjection of recombinant X-PAK1-Cter active kinase into progesterone-treated oocytes prevents c-Mos accumulation and activation of both MAPK and maturation-promoting factor (MPF). In conditions permissive for MAPK activation, MPF activation still fails. We demonstrate that a constitutive truncated version of X-PAK1 (X-PAK1-Cter) does not prevent the association of cyclin B with p34(cdc2) but rather prevents the activation of the inactive complexes present in the oocyte. Proteins participating in the MPF amplification loop, including the Cdc25-activating Polo-like kinase are all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. Our results indicate that X-PAK and protein kinase A targets in the control of oocyte maturation are similar and furthermore that this negative regulation is not restricted to meiosis, because we demonstrate that G2/M progression is also prevented in Xenopus cycling extracts in the presence of active X-PAK1.     

YKL-40, a mammalian member of the chitinase family, is a matrix protein of specific granules in human neutrophils

YKL-40, also called human cartilage glycoprotein-39 (HC gp-39), is a member of family 18 glycosyl hydrolases. YKL-40 is secreted by chondrocytes, synovial cells, and macrophages, and recently it has been reported that YKL-40 has a role as an autoantigen in rheumatoid arthritis (RA). The function of YKL-40 is unknown, but the pattern of its expression in normal and disease states suggests that it could function in remodeling or degradation of the extracellular matrix. High levels of YKL-40 are found in synovial fluid from patients with active RA. Neutrophils are abundant in synovial fluid of patients with RA, and the cells are assumed to play a role in joint destruction in that disorder. Therefore, we examined whether neutrophils are a source of YKL-40. YKL-40 was found to colocalize and comobilize with lactoferrin (the most abundant protein of specific granules) but not with gelatinase in subcellular fractionation studies on stimulated and unstimulated neutrophils. Double-labeling immunoelectron microscopy confirmed the colocalization of YKL-40 and lactoferrin in specific granules of neutrophils. Immunohistochemistry on bone marrow cells showed that neutrophil precursors begin to synthesize YKL-40 at the myelocyte-metamyelocyte stage, the stage of maturation at which other specific granule proteins are formed. Assuming that YKL-40 has a role as an autoantigen in RA by inducing T cell-mediated autoimmune response, YKL-40 released from neutrophils in the inflamed joint could be essential for this response. In RA and other inflammatory diseases, YKL-40 released from specific granules of neutrophils may be involved in tissue remodeling or degradation.     

A 50 kilodalton protein associated with raf and pp60(v-src) protein kinases is a mammalian homolog of the cell cycle control protein cdc37

Several oncogenic protein kinases including c-raf-1 and pp60(v-src) are known to directly interact with the 90 kDa heat shock protein (hsp90)/p50 complexes. Using a monoclonal antibody to detect p50 during a purification scheme, p50 was purified to homogeneity. Internal amino acid sequence information was obtained and used to clone a partial cDNA. Comparison of the p50 sequence to other cloned proteins revealed 89% homology with a glycosaminoglycan-binding protein and 54% homology with Drosophila cell cycle control protein (cdc) 37. Monoclonal and polyclonal antibodies were produced against a cleaved fusion protein that recognizes p50 with a high level of specificity. These antibodies recognize the 50 kDa protein present in c-raf-1 and pp60(v-src) complexes. No other proteins were recognized with these antibodies suggesting that p50 is a unique protein. Immunocytochemical visualization of p50 in NIH 3T3 cells indicates a primarily cytoplasmic localization around the nuclear membrane. A survey of p50 expression in murine tissues on a protein blot revealed the following relative levels of expression; thymus > spleen > brain > heart > kidney > liver > lung > skeletal muscle. These results link studies demonstrating complexation of certain kinases with hsp90/p50 in mammalian cells and a number of reports in yeast and Drosophila, demonstrating the importance of cdc37 in cell cycle and kinase function.     

A cyclin-dependent kinase family member (PHOA) is required to link developmental fate to environmental conditions in Aspergillus nidulans

We addressed the question of whether Aspergillus nidulans has more than one cyclin-dependent kinase gene and identified such a gene, phoA, encoding two PSTAIRE-containing kinases (PHOAM1 and PHOAM47) that probably result from alternative pre-mRNA splicing. PHOAM47 is 66% identical to Saccharomyces cerevisiae Pho85. The function of this gene was studied using phoA null mutants. It functions in a developmental response to phosphorus-limited growth but has no effect on the regulation of enzymes involved in phosphorus acquisition. Aspergillus nidulans shows both asexual and sexual reproduction involving temporal elaboration of different specific cell types. We demonstrate that developmental decisions in confluent cultures depend upon both the initial phosphorus concentration and the inoculation density and that these factors influence development through phoA. In the most impressive cases, absence of phoA resulted in a switch from asexual to sexual development (at pH 8), or the absence of development altogether (at pH 6). The phenotype of phoA deletion strains appears to be specific for phosphorus limitation. We propose that PHOA functions to help integrate environmental signals with developmental decisions to allow ordered differentiation of specific cell types in A.nidulans under varying growth conditions. The results implicate a putative cyclin-dependent kinase in the control of development.     

A mild phenotype associated with der(9)t(3;9) (p25;p23)

A female infant is described with hypoglycaemia, hypotonia, obesity of the trunk and thighs, and mild dysmorphic features. Growth parameters were consistently above the 90th centile. Chromosome analysis showed her to have a derived chromosome 9 inherited from a maternal t(3;9)(p25;p23) by adjacent I segregation. She had features in common with both the dup(3p) and del(9p) syndromes. There are few reports of this chromosome rearrangement and the features are milder than expected for the degree of imbalance, complicated in males by sex reversal. The repeated reports of macrosomia may suggest an overgrowth syndrome.