Regulation of acid phosphatase activity in human promyelocytic leukemic cells induced to differentiate in culture.

Induction of differentiation of a human promyelocytic leukemic cell line (HL60) in culture is accompanied by changes in acid phosphatase (Acpase) activity. The increase in activity is less than twofold when the leukemic cells are stimulated by dimethylsulfoxide (DMSO) to differentiate into metamyelocytes and granulocytes but is eightfold when the cells are stimulated by the tumor-promoting agent 12-0-tetradecanoylphorbol 13-acetate (TPA) to differentiate into macrophage-like cells. Five different isozymes of Acpase were separated by acrylamide gel electrophoresis. Isozyme 1, the most anodal isozyme, was found to be present in undifferentiated, DMSO-treated and TPA-treated cells; isozyme 2 was a very faint band observed both in DMSO- and TPA-treated cells, the isoenzymes 3a and 3b were present only in TPA-induced cells; and isozyme 4, the most cathodal isozyme, was present both in TPA- and DMSO-induced cells. A time sequence study on the appearance of the various forms after TPA treatment indicated that the expression of the isozymes is regulated in an uncoordinated fashion. Acpase activity has been shown by ultrastructural cytochemistry to be localized in the entire rough endoplasmic reticulum (RER) and in areas of the smooth endoplasmic reticulum (SER) located near the Golgi complex in differentiating cells but to be extremely weak, if at all detectable, in undifferentiated promyelocytes.

The mepsMAP server. Mapping epitopes on protein surface: mining annotated proteins.

For a growing number of biologists DNA or protein data are typically retrieved and managed on the Web, and not in the laboratory. A large number of bioinformatics datasets from primary and (thousands of) secondary databases are scattered on the Web in various formats. A biologist end-user might need to access and use tens of databases and tools every day. For this reason, the bioinformatics community is developing more and more service-oriented architectures (SOAs): software architecture of loosely coupled software services that can be accessed without knowledge of, or control over, their internal architecture. Data-processing and analysis tasks can be automated by having free access to bioinformatics Web services (WSs) that are the building blocks of the SOAs. In this paper we introduce a new bioinformatics Web server, mepsMAP (mapping epitopes on protein surface: Mining Annotated Proteins), developed to identify the recognition sites between antibodies and their cognate antigens. In some cases, the recognition site is represented by a continuous segment of the antigen sequence, but much more often the epitope is "conformational," i.e., the antibody recognizes the location and type of exposed antigen side chains that are not necessarily contiguous in the antigen's sequence, but brought together by its three-dimensional structure. A facility on the server allows the user to search putative conformational epitopes on protein surface, querying the system for proteins with a given annotation. The mepsMAP server has been implemented as a SOA composed by a database and a set of four WSs. We present here the software architecture of the system with a detailed description of the WS dataflow that has been optimized to provide the best computing performance while maintaining the easiest end-user access to the system via a Web interface.

Differential expression of the globin genes in human leukemia K562(S) cells induced to differentiate by hemin or butyric acid.

Human leukemia K562(S) cells were induced to differentiate by 50 microM hemin or 1.4 mM butyric acid, and the types of hemoglobins synthesized were compared. In both cases, embryonal hemoglobins [Portland, Gower 1, Hb X, and fetal hemoglobin (Hb-F)] were detected. Butyric acid-treated K562(S) cells contained mostly Hb Gower 1 (zeta 2 epsilon 2) and a hemoglobin with the electrophoretic characteristics of Portland (gamma 2 zeta 2). For hemin-treated K562(S), the most abundant hemoglobin synthesized by Hb X (epsilon 2 gamma 2), and the second most abundant was Bart's (gamma 4). Traces of Gower 1 were observed in nontreated K562(S) cells. The kinetics of hemoglobin induction as a result of the two treatments differed; increased hemoglobin synthesis was detected after only 24 hr of hemin treatment, whereas 4 days were required in butyric acid-treated cells. Both hemin and butyric acid were able to induce their respective patterns of hemoglobin synthesis independent of the presence of serum in the K562(S) growth medium. Analysis of the globin chains in induced K562(S) cells induced to differentiate indicated that, with both inducers, adult alpha- but not beta-globin chains were present. Karyotype analysis of K562(S) cells revealed a nearly triploid chromosome complement with a modal number of 68 chromosomes. Three copies of chromosome 11 and four copies of chromosome 16 (coding for the beta-like and alpha-like globin genes, respectively) were present. A large marked chromosome, involving chromosome 7, and a Philadelphia chromosome were also seen. These data characterize the K562(S) subline and also indicate that hemin and butyric acid differ in their effects on the expression of embryonal globin genes.

Che-1 modulates the decision between cell cycle arrest and apoptosis by its binding to p53.

The tumor suppressor p53 is mainly involved in the transcriptional regulation of a large number of growth-arrest- and apoptosis-related genes. However, a clear understanding of which factor/s influences the choice between these two opposing p53-dependent outcomes remains largely elusive. We have previously described that in response to DNA damage, the RNA polymerase II-binding protein Che-1/AATF transcriptionally activates p53. Here, we show that Che-1 binds directly to p53. This interaction essentially occurs in the first hours of DNA damage, whereas it is lost when cells undergo apoptosis in response to posttranscriptional modifications. Moreover, Che-1 sits in a ternary complex with p53 and the oncosuppressor Brca1. Accordingly, our analysis of genome-wide chromatin occupancy by p53 revealed that p53/Che1 interaction results in preferential transactivation of growth arrest p53 target genes over its pro-apoptotic target genes. Notably, exposure of Che-1(+/-) mice to ionizing radiations resulted in enhanced apoptosis of thymocytes, compared with WT mice. These results confirm Che-1 as an important regulator of p53 activity and suggest Che-1 to be a promising yet attractive drug target for cancer therapy.

Sequence comparison of human and murine erythrocyte alpha-spectrin cDNA.

The results of hybridization analyses using cDNA probes for mouse and human alpha-spectrin mRNA indicate that a single gene encodes the alpha-subunit of erythrocyte spectrin. Sequencing of the cDNA clones showed that they code for 370 amino acids (aa) covering three repeat domains close to the C terminus of alpha-spectrin. The cloned cDNAs will now permit the isolation of the alpha-spectrin gene and should lead to the characterization of the genetic aspects in human hereditary anemias in which alpha-spectrin has been characterized as the site of the molecular defect.

Computer-assisted analysis of fetal movements in intrauterine growth retardation (IUGR).

A quantitative analysis of various fetal activities (mouth, eye and gross body movements) was made in 10 IUGR human fetuses. The aim of the study was to see whether IUGR fetuses move differently to normal fetuses. Each real-time ultrasound recording lasted 1 h and the analysis of various activities was carried out during replay of video recordings by means of a specially designed computer program. The following aspects have been investigated: (1) incidence, duration and interval for each of the fetal activities described; (2) the relationship between incidence, duration and interval for each single activity; (3) the correlations between the different activities. The results were compared with a group of 10 fetuses from normal pregnancies. On quantitative evaluation no clear effects due to uncomplicated IUGR could be detected except for median duration of eye movements, which turned out to be longer in the IUGR group. The evaluation of correlations between the characteristics (incidence, duration and interval) of each activity showed a positive correlation between incidence and duration of mouthing movements in the IUGR group, not found in the normal group. The study of the correlation between different fetal activities has shown an inverse correlation between mouthing and other activities in the normal fetuses, not found in the IUGR group. We conclude that in mildly affected fetuses with no evidence of hypoxia, there are no quantitative differences compared to normal fetuses in terms of the motility studied. The only differences found were in relation to the performance of such activities and they could reflect a dysfunction of the central nervous system resulting from a metabolic disturbance.

Behavior of a cell line derived from a mouse submaxillary adenocarcinoma during the initial 480 days in vitro.

A cell line was established from a transplantable adenocarcinoma, containing viral particles of the A and B type, drived from a tumor appearing spontaneously in the submaxillary region of a male mouse of the C3H/He strain. This line, after 480 days in vitro, did not change the original epithelial-like morphology, the viral expression, the membrane immunofluorescence and the degree of agglutination by various plant lectins. After 208 days of culture, the presence of up to 3 pairs of metacentric chromosomes appeared in about 55% of the cells. However, this change in the chromosomal pattern was not sufficient, at least within the limits of our observation, to modify significantly the other parameters investigated, with the possible exception of the oncogenicity, which showed a modest decrease after 296 days of culture.

[Behavioral states. An in utero study]. / Stati comportamentali. Studio in utero.

OBJECTIVE: This study aims to show how a single linear transducer and different state variables can be used to enable accurate identification of behavioural states. DESIGN: Prospective observation study. SETTING: Clinic of Obstetrics and Gynaecology III at the "Federico II" University of Naples. SUBJECTS: Fifteen fetuses of women hospitalized in the Clinic of Gynaecology at the "Federico II" University of Naples at the end of a full-term, uncomplicated, single pregnancy with a reliably dated last menstruation. METHODS: Ultrasound observation, registration and data processing of various fetal activities in the uterus mouth movements (sucking), other mouth movements, eye movements, gross body movements. RESULTS: The fetal activities identified correspond exactly to the criteria established for the definition of state variables. An examination of the data thus obtained shows an inverse correlation between mouth movements and the other activities, and a direct correlation between the other three variables we considered. CONCLUSIONS: The results obtained in our study show that this method can be profitably used to identify the behavioural states, in full-term, uncomplicated pregnancies.

A critical analysis of the anterior-posterior radiographic anatomy of the ankle syndesmosis.

To determine the normal anatomic radiographic land-marks of the ankle syndesmosis, standardized anterior-posterior radiographs of the right ankle were performed on 40 male and 40 female volunteers. The average tibiofibular clear space was 3.8 mm in females, 4.6 mm in males, and 4.2 mm overall. The tibiofibular overlap measured 6.0 mm in females, 9.6 mm in males, and 7.8 mm overall. Due to this variability and the gender differences, we investigated the anatomy of the syndesmosis as ratios of the potentially variable values to fixed landmarks. The ratio of the tibiofibular overlap to the fibular width averaged 54% and the ratio of the tibiofibular clear space to the fibular width averaged 30%, with no statistically significant difference due to gender. Our data show that for 90% prediction intervals, the values are: (1) tibiofibular clear space less than 5.2 mm in women and 6.5 mm in men; (2) tibiofibular overlap of greater than 2.1 mm in females and 5.7 mm in males; (3) tibiofibular overlap:fibular width ratio greater than 24%; (4) tibiofibular clear space:fibular width ratio less than 44%. Additionally, using a linear regression model, a prediction of the tibiofibular overlap can be made when using the distance (in millimeters) from the incisura fibularis to the lateral tibial (LT) border: tibiofibular overlap = 0.862 x lateral tibia - 2.62 (P = .0001). Utilization of these values will help in the determination of posttraumatic disruption of the syndesmosis and postoperative assessment of mortise reduction.

Functionalized halloysite multivalent glycocluster as a new drug delivery system.

A new design for halloysite nanotube materials was obtained by grafting chemically modified cyclodextrin units onto the nanotube surface. In particular, grafted cyclodextrins were decorated with thiosaccharide pendants, in order to mimic the well-known binding of sugars to proteins and the glyco-cluster effect occurring during cellular recognition events. The obtained materials were characterized by using a combination of varied techniques (FT-IR spectroscopy, thermogravimetric analysis, scanning electron microscopy, dynamic light scattering, turbidimetry), and their potential drug-delivery abilities were tested by studying their interactions with the common naturally occurring anticancer agent curcumin. A suitable model describing the interaction between our materials and curcumin is proposed.

Modulation of hemoglobin synthesis in K-562(S) cells treated with interferons.

Treatment of K-562(S) cells with human interferons (HulFN) alpha, beta, or gamma results in a modulation of hemoglobin (Hb) production. When K-562(S) cells, "induced" with butyric acid or hemin, are given low dosages of all three types of IFN, the percent benzidine-positive cells doubles. Treatment with low doses of IFN causes the acceleration and increased production of those Hb types (as shown by Cellogel analysis) that are already synthesized either in untreated or in "induced" cultures. These results were confirmed by using pure HulFN-beta, and were abrogated, for HulFN-alpha, in presence of its specific antiserum. In contrast, cultures of K-562 cells treated with either inducer and given more than 10(4) IU/ml of alpha- or beta-IFN show a dose-dependent decrease of hemoglobinization in the absence of cell death. The inhibitory effect was reversible upon removal of IFN and culture reseeding in IFN- and inducer-free medium. The significance of both sets of data is strengthened by the pronounced heterogeneity of K-562S cells with respect to their sensitivity to IFN treatment as evaluated by the establishment of the antiviral state. Apparently, one cell out of two is sensitive to IFN, which suggests that the magnitude of IFN effects described here may be larger than it appears to be from the data taken at face value.

Modulation by cell trypsinization of Sendai virus expression in African green monkey kidney cells: first infection and establishment of a carrier state.

Two African green monkey kidney (AGMK) cell lines, 37RC (interferon-producing) and Vero (non-interferon-producing), were infected by egg-grown Sendai virus passaged in eggs at high and low m.o.i. The appearance of haem-adsorption, and cytopathic effect (c.p.e.) as well as the presence of haemagglutinating virions in the supernates were much more pronounced with a virus seed obtained with 10(-3) diluted passages than with a seed obtained with undiluted inoculum. They were also independent of interferon production (data obtained in 37RC and Vero cells were almost superimposable). In studies carried out with the virus seed prepared at high dilution the establishment of infection was maximal when monolayers were infected as soon as 5 h after trypsinization and cell seeding, regardless of cell density. Virus in supernates obtained from cultures infected 5, 20 or 50 h after seeding exhibited a greatly reduced infectivity for monkey cells, but not apparently for chick embryos. Trypsin treatment of the virus supernates restored their infectivity for AGMK cells more efficiently for virus released from cells infected 5 h after seeding than for virus released from cells infected later after seeding. In keeping with these observations, virus in supernates from cultures infected 5, 20 or 50 h after seeding contained increasing amounts of auto-interfering virions. The decreased infectivity obtained in cell supernates was not accounted for by major differences in virus RNA synthesis. Similarly, the optimum infection established in cells seeded only for 5 h was not due to increased virus adsorption. Several lines of cells surviving first infection with egg-grown Sendai virus have been obtained and kept in cultures for 3 to 18 months. Virus release and c.p.e. apparently become reduced in the cells surviving the first infection until the newly repopulated monolayers must undergo trypsinization. Weekly protease treatments of the cells reactivate all parameters of virus infection which again will tend to disappear slowly and then reappear following each trypsinization ('intermittent' carrier state). The establishment and the 'intermittent' reactivation of these lines were not prevented by the inclusion in the medium of anti-Sendai virus serum. Temperature-sensitive ts functions do not seem to play an important role in this virus-host relationship.

Anti-idiotypic antibodies to delineate epitope specificity of anti-amiloride antibodies.

Amiloride and related compounds have found widespread use as cation transport inhibitors. We have previously raised a series of polyclonal anti-amiloride antibodies using different amiloride-protein conjugates as immunogens, where amiloride was coupled to protein either through its guanidino moiety or through its 5-aminopyrazinyl moiety. The anti-amiloride antibodies recognized distinct sites on amiloride, and the site of attachment of amiloride to carrier protein was a critical factor in determining which part of the amiloride molecule was recognized by the anti-amiloride antibody. The specificity of binding of amiloride analogues to these polyclonal anti-amiloride antibodies mimicked the specificity of binding of amiloride analogues to selected isoforms of the epithelial Na+ channel or the Na+/H+ exchanger, suggesting that antigen binding site of these antibodies might be similar in structure to amiloride binding sites on selected Na+ transport proteins. We previously generated monoclonal anti-idiotypic antibodies RA2.4 and RA6.3 by an auto-anti-idiotypic approach, using amiloride coupled to albumin through the guanidinium moiety (amiloride-A1). We have now raised a series of monoclonal anti-idiotypic antibodies, T6, T26, T40, and T181, using amiloride coupled to keyhole limpet hemocyanin through the 5-aminopyrazinyl moiety (amiloride-A5) as an immunogen with the same auto-anti-idiotypic approach. These monoclonal anti-idiotypic antibodies recognized both polyclonal anti-amiloride-A1 and anti-amiloride-A5 antibodies, suggesting that idiotype-anti-idiotype interaction was not epitope restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA.

A rabbit monospecific antibody for mouse beta-spectrin was used to screen a mouse anemic spleen cDNA expression library. A mouse beta-spectrin cDNA clone was isolated and identified by its ability to make mouse beta-spectrin-like antigens in Escherichia coli. This clone was used to probe total RNA from various mouse tissues. Anemic spleen RNA showed two strongly hybridizing RNA species of approximately 6 and 8 kb. Two very faintly hybridizing bands of about 6 kb and 10 kb could also be seen in total mouse brain RNA. All of these bands could be detected after hybridization under both stringent and nonstringent conditions. This suggests that erythroid beta-spectrin may also be expressed in the brain. No bands could be detected in kidney, liver, or spleen RNA. Southern blot analysis of mouse genomic DNA showed a single hybridizing band after digestion with several restriction endonucleases even under nonstringent conditions. Nucleotide sequencing of the cDNA insert revealed almost complete identity between the N-terminus of the deduced amino acid sequence of the cDNA clone and the C-terminal 15 amino acids of a peptide derived from the beta-8 repeat unit of human erythrocyte beta-spectrin. The deduced amino acid sequence contained most of the conserved amino acids characteristic of the 106 amino acid repeat unit first found in human alpha-spectrin and thus provides the first evidence for a complete 106 amino acid repeat unit structure in beta-spectrin.

Surface phenotype of clonogenic cells in acute myeloid leukemia defined by monoclonal antibodies.

Colony-forming cells in ten cases of acute myeloid leukemia (AML) were studied with six cytotoxic monoclonal antibodies that react with antigens expressed at discrete stages of differentiation of normal and leukemic hematopoietic cells. The reactivity of the whole leukemic population was measured by indirect immunofluorescence, and the reactivity of the colony-forming cells was established by complement-mediated cytotoxicity and by fluorescence activated cell sorting. Comparison of the immunofluorescent reactivity with cytotoxicity and cell sorting showed that colony-forming cells were found within a fraction of the leukemic subpopulations that expresses these antigens. This finding implies that immunofluorescence reactivity of the total leukemic population does not necessarily predict the phenotype of the clonogenic cells. When the surface phenotype of the clonogenic leukemic cells was compared to that previously established for normal marrow hemopoietic clonogenic cells, several patterns were seen: (1) in four of ten cases, the clonogenic cells expressed a phenotype like that of relatively mature normal granulocyte-macrophage colony-forming cells (late CFU-GM) or, (2) in two cases, a phenotype similar to the less mature colony-forming cells (early CFU-GM or CFU-GEMM), and (3) in four cases, a composite phenotype of early and late CFU-GM. Thus, the level of impairment of differentiation in AML may vary from case to case. In those cases phenotypically similar to the late CFU-GM, it may be possible to separate leukemic clonogenic cells from less mature normal clonogenic cells using monoclonal antibodies selectively cytotoxic for the late CFU-GM.

Outpatient uterine artery embolization for symptomatic uterine fibroids: experience in 49 patients.

PURPOSE: To assess the feasibility of performing uterine artery embolization as an outpatient treatment for symptomatic uterine fibroids. MATERIALS AND METHODS: Forty-nine consecutive patients (mean age, 44.5 years; range, 28-54 years) underwent uterine artery embolization during a 12-month period. Embolization was performed with 350-500 microm polyvinyl alcohol particles (44 of 49) or Gelfoam pledgets (five of 49). At discharge, patients were given instructions regarding the constitutional symptoms to expect after embolization. A specific medication regimen consisting of prochlorperazine, ketorolac, meperidine, and hydrocodone was prescribed for relief of these symptoms. All patients were telephoned within 24 hours of discharge. During long-term follow-up, a questionnaire was administered to all patients to evaluate the periprocedural experience. Three-month clinical follow-up was available in 26 patients and 6-month imaging follow-up was available in 16 patients. RESULTS: Fourteen patients presented with menorrhagia, six had bulk-related symptoms (abdominal distension, stress incontinence, pelvic pain), and 29 had both. Technical success for bilateral embolization was 98%. Forty-seven of 49 patients were discharged to home 6-8 hours after the procedure; two patients required overnight observation in an ambulatory unit (one because of postprocedure hypertension and one because of a late procedure completion time). At the first follow-up phone call, reported symptoms included pelvic pain/cramping in 83.7% (41 of 49), fatigue in 75.5% (37 of 49), nausea/vomiting in 46.9% (23 of 49), and a nonpurulent vaginal discharge in 18.4% (nine of 49). These symptoms were satisfactorily controlled with discharge medications in 48 of 49 patients. No patients returned to the hospital or visited an emergency room during the first 48 hours after discharge. Forty-six of 49 patients were satisfied with the decision for home discharge. The average uterine volume reduction in 16 patients with 6-month imaging follow-up was 47.5%; 88.5%, of patients (23 of 26) with 3-month clinical follow-up reported improvement or elimination of symptoms. CONCLUSION: With defined telephone follow-up, staff availability, and a protocol designed to alleviate the postprocedure constitutional symptoms, uterine artery embolization is both safe and effective when performed as an outpatient procedure.

Differentiation of human leukemias in response to 12-0-tetradecanoylphorbol-13-acetate in vitro.

Leukemic cells from patients with acute myeloid leukemia underwent morphological, functional, and histochemical changes within 24-48 hr after treatment with 1.6 x 10-18 M 12-0-tetradecanoylphorbol-13-acetate (TPA). The changes included adhesion to the plastic substrate, a 4-6-fold increase in the number of phagocytic cells, and an increase in the number of alpha-naphthyl-acetate esterase (alpha-NAE) positive cells. In contrast, TPA treatment of cells from patients with acute lymphoblastic leukemia caused some aggregation of cells in suspension, but no changes in adhesion, phagocytosis, or alpha-NAE. Of the four cases of undifferentiated or unclassified leukemias studied, two failed to respond to TPA, one responded with a myeloid (adhesion) pattern, and one with a lymphoid (aggregation) pattern. These data suggest that leukemic myeloblasts retain the ability to express a variety of differentiated functions, and in some cases, it may be possible to use TPA as a tool to test the differentiative potential of undifferentiated human leukemias.

Effects of a chromatin low molecular weight peptidic fraction on differentiation markers and virus production in Friend leukemia cells.

Low molecular weight chromatin peptides exert a dose-dependent inhibition of Dimethylsulfoxide (DMSO)-induced erythroid differentiation of murine Friend Leukemia Cells (FLC). This effect correlates with the degree of purification of the peptide fractions. Crot analysis of globin mRNA amounts in DMSO-treated FLC given the peptides showed a 4-5-fold decrease of messenger RNA in the cytoplasma with no nuclear storage of globin transcripts. Spectrin accumulation in "induced" FLC is inhibited as well. The effects of the peptides on erythroid markers are reversible upon removal of the compounds. They also appear to be specific for induced gene expression as (1) no effects are observed on cell growth and RNA synthesis in normal non-differentiating cell lines; and (2) no changes have been detected with regard to the expression of integrated viral genes coding for continuous shedding of viral particles.

Can the administration of antithrombin III decrease the risk of cerebral hemorrhage in premature infants?

This study was carried out to determine whether the administration of antithrombin III decreases the risk of intraventricular hemorrhage in premature infants. In a randomized study, 60 infants born before 30 weeks of gestation were assigned to receive a loading dose of antithrombin III or placebo. There was no significant difference in the incidence of intraventricular hemorrhage between the antithrombin III and the placebo group (27.5 vs. 32%). Partial thromboplastin time, Quick's prothrombin time and platelet count were also not significantly different between the 2 groups. We conclude that the administration of antithrombin III during the first 2 days of life does not decrease incidence of intraventricular hemorrhage. Antithrombin III is a very expensive therapy and its benefits should be carefully investigated before being recommended as valuable therapy.