Growth factor-mediated tumor cell proliferation in hairy cell leukemia.

Leukemic B cells from seven patients with hairy cell leukemia (HCL), six of which contained the Tac antigen, were assayed in vitro for growth factor-mediated cell proliferation. The HCL cells showed typical phenotypic profiles by monoclonal antibody analysis. The tumor cells, which do not grow spontaneously in vitro, were found to proliferate in all but one case in response to partially purified B cell growth factor (BCGF) without anti-mu or Sac activation. Recombinant interleukin 2 however produced only a marginal response and could not support leukemic cell growth in vitro. BCGF, however, did stimulate in vitro cell growth and supported the establishment of continuous (greater than 60 d in vitro) in four of the seven HCL cases.

A comparison of the loosely bound and tightly bound nonhistone proteins from Ehrlich ascites tumor chromatin.

The nonhistone proteins of Ehrlich ascites tumor chromatin have been separated into a loosely bound and two tightly bound protein fractions by sequential extraction of chromatin with 0.35 M NaCl and 2 M NaCl:5 M urea. The nonhistone proteins thus obtained were examined for their chemical composition and distribution of DNA polymerase, RNA polymerase, and protein kinase activities. In addition, the effect of these nonhistone proteins on transcription of DNA in vitro has been determined. The results indicate that these nonhistone proteins, fractionated on the basis of their extractability, exhibit varied compositional characteristics and play different functional roles in the synthesis of DNA and RNA and in the possible control of gene activity.

Purification of a low molecular weight non-histone chromosomal protein.

A non-histone chromosomal proteins was extracted from rat liver chromatin with 0.35 M NaCl and purified more than 2758 times to near homogeneity by hydroxyapatite, gel filtration, and phosphocellulose chromatography. The final fraction was greater than 95% pure as judged by non-denaturing gel electrophoresis. The protein, designated loosely bound non-histone chromosomal protein 1, had an observed molecular weight of 15 700. This protein was demonstrated to increase the amount of RNA synthesized in a heterologous (Escherichia coli RNA polymerase) transcription system and, therefore, this activity was also used to monitor its purification. The availability of highly purified loosely bound non-histone chromosomal protein 1 will make possible an examination of its structural and/or functional role in chromatin.

Prototypical HTLV-I/II infection is rare in LGL leukemia.

The etiology of LGL leukemia is not known; however, we recently detected HTLV-II in a patient with LGL leukemia. In this study, we found that sera from 6 of 28 patients with LGL leukemia were positive for HTLV-I/II using a whole virus ELISA; moreover, the ELISA-negative sera were near the positive cut-off value. Therefore, we performed additional studies on these sera using commercially available assays which can confirm and distinguish HTLV-I from HTLV-II infection. Serum from only one patient was confirmed positive using conventional criteria (HTLV-II+). Sera from 25 patients (89%) had indeterminate reactivity on Western blot assays. Of these, sera from 21 (84%) reacted to gag protein p24; 12 (48%) reacted with recombinant env protein p21e, and 10 (40%) reacted with both. We could not detect HTLV-I/II pol or pX gene sequences in these patients using polymerase chain reaction analyses, with the exception of the HTLV-II-infected patient described previously. These data show that most patients with LGL leukemia are not infected with prototypical HTLV-I or HTLV-II. The frequent reactivity of patient sera to HTLV-I/II gag protein p24 and to env protein p21e, however, suggests that a deleted or variant form of HTLV-I/II may be associated with LGL leukemia.

Comparison of transcription stimulating, phenol-soluble non-histone chromosomal proteins in normal rat liver and transplantable hepatocellular carcinomas.

The non-histone chromosomal proteins (NHCP) of a rapidly and slowly proliferating transplantable hepatocellular carcinoma (THC) were compared to those of normal and regenerating rat liver. The total quantity of NHCP is approximately threefold higher in the THCs than in either normal rat liver at 4 h and 44 h regenerating rat liver. Only those NHCP that can be extracted from chromatin by 0.35 M NaCl were further examined and it was observed that the proteins of this highly complex fraction could be further fractionated by their differential phenol-solubility. The phenol-soluble 0.35 NHCP contained protein(s) capable of stimulating the level of DNA-directed RNA synthesis in vitro. The total amount of this stimulatory activity was 5 times higher in the rapidly growing THC and 1.6 times higher in the slowly growing THC than in normal rat liver. In order to assess the contribution of cell-cycle dependent alterations on the increase in the amount of stimulatory activity in the THCs, 44 h regenerating rat livers were examined. This tissue represents a mix of cells in various stages of the cell cycle which is similar to that found in the THCs. It was found that the total quantity of NHCP in the 44 h regenerating rat liver was the same as in normal rat liver. The total amount of the stimulatory activity also was similar in both the normal and 44 h regenerating rat liver. The amount of the stimulatory activity was found to double in 4 h regenerating rat liver, however. These data suggest that the alterations observed in the NHCP of the THCs are not due solely to cell cycle dependent changes, but may represent malignancy dependent alterations.

Chromatin conformation modulates repair of single strand interruptions by polynucleotide ligase-[3H] AMP.

Conformationally distinct chromatin populations were utilized as substrates to quantitate the relative amount of and accessibility of internal 5'-phosphomonoester breaks in DNA-chromatin. In these studies, a constant amount of chromatin as well as deproteinized DNA derived from the respective chromatin sample was titrated with increasing quantities of adenylated polynucleotide ligase intermediate. This enzyme intermediate releases its AMP moiety while repairing a DNA single strand interruption, release of AMP being directly proportional to the number of internal 5'-phosphomonoester breaks repaired. Results of this study indicate that the ability of polynucleotide ligase to repair DNA breaks within chromatin was affected by the conformational state of the chromatin. The degree of conformational constraint present in a given chromatin, therefore, determined the capacity of the enzyme to repair internal DNA 5'-phosphomonoester breaks.

Monoclonal antibody identifies a highly conserved and immunodominant epitope of the human immunodeficiency virus transmembrane protein.

A murine monoclonal antibody (MAb), 10E9, has been generated which identifies a conserved and immunodominant epitope of the human immunodeficiency virus (HIV) transmembrane protein, gp41. The MAb reacts with the protein backbone of the mature env gene product and also with polyprotein precursor, gp160. Human sera were tested for their ability to competitively inhibit the immunoreactivity of MAb 10E9. Of 100 serum samples obtained from patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC), all showed strong inhibition to the reaction. In contrast, sera obtained from normal donors or those with other viral infections failed to perturb the binding activity of MAb 10E9. The geographic diversity of the AIDS/ARC patients studied provides evidence that the 10E9 epitope of gp41 is highly conserved.

Biomarker discovery by plasma proteomics in familial Brugada Syndrome.

Brugada Syndrome (BS) is a polygenic inherited cardiac disease characterized by life-threatening arrhythmias and high incidence of sudden death. In this study, two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (LC-MS/MS) was used to investigate specific changes in the plasma proteome of BS patients and family members sharing the same gene mutation (SCN5AQ1118X), with the aim to identify novel disease biomarkers. Our data demonstrate that the levels of several proteins were significantly altered in BS patients compared with controls. In particular, apolipoprotein E, prothrombin, vitronectin, complement-factor H, vitamin-D-binding protein, voltage-dependent anion-selective channel protein 3 and clusterin were considerably increased in plasma sample of BS patients, whereas alpha-1-antitrypsin, fibrinogen and angiotensinogen were considerably decreased; moreover, post-translational modifications of antithrombin-III were detected in all affected individuals. On the light of these results, we hypothesize that these proteins might be considered as potential markers for the identification of disease status in BS.

Dynamic simulations of pathways downstream of ERBB-family, including mutations and treatments: concordance with experimental results.

The pathways downstream of ErbB-family proteins are very important in BC, especially when considering treatment with onco-protein inhibitors. We studied and implemented dynamic simulations of four downstream pathways and described the fragment of the signaling network we evaluated as a Molecular Interaction Map. Our simulations, enacted using Ordinary Differential Equations, involved 242 modified species and complexes, 279 reversible reactions and 111 catalytic reactions. Mutations within a single pathway tended to be mutually exclusive; only inhibitors acting at, or downstream (not upstream), of a given mutation were active. A double alteration along two distinct pathways required the inhibition of both pathways. We started an analysis of sensitivity/robustness of our network, and we systematically introduced several individual fluctuations of total concentrations of independent molecular species. Only very few cases showed significant sensitivity. We transduced the ErbB2 over-expressing BC line, BT474, with the HRAS (V12) mutant, then treated it with ErbB-family and phosphorylated MEK (MEKPP) inhibitors, Lapatinib and U0126, respectively. Experimental and simulation results were highly concordant, showing statistical significance for both pathways and for two respective endpoints, i.e. phosphorylated active forms of ERK and Akt, p one tailed = .0072 and = .0022, respectively. Working with a complex 39 basic species signaling network region, this technology facilitates both comprehension and effective, efficient and accurate modeling and data interpretation. Dynamic network simulations we performed proved to be both practical and valuable for a posteriori comprehension of biological networks and signaling, thereby greatly facilitating handling, and thus complete exploitation, of biological data.

Increasing smoking cessation counseling by advanced practice nurses.

The magnitude of individual and societal problems caused by tobacco use mandates that all primary care providers identify and advise smokers to quit. However, this topic has received little attention in the nurse practitioner literature. The purpose of this project is to identify effective methods by which advanced practice nurses can increase the identification and counseling of smokers by reviewing research on this topic. The articles for review were obtained through a computerized literature search and a review of related reference lists. The articles were analyzed and categorized into three groups: office-wide interventions to increase provider identification and counseling of smokers, smoking cessation training programs for providers, and studies using the stages of change theory. Provider smoking cessation programs and office-wide reminders increased the identification and counseling of patients who smoke. The stages of change theory helped explain the steps smokers must progress through to cease smoking. Interventions appropriate for various stages in the cessation process are suggested.

[Anticoagulation in atrial fibrillation: not yet settled]. / Tratamiento anticoagulante en fibrilación auricular: aún no está todo resuelto.

A great deal of interest has received atrial fibrillation, the most common arrhythmia in adults, due to its complications and difficult treatment its most dreaded complication is atrial thrombi formation with the subsequent risk of embolization. There are several reports defining risk factors for embolic complications and the usefulness of anticoagulants for their prevention. We review the state of the art of anticoagulation in atrial fibrillation not associated to rheumatic valvulopathy. We also give tools to assess embolic risk and to determine the anticoagulant choice for the different presentation forms of atrial fibrillation.

[Prevalence of dental fluorosis in Chile: a pilot study]. / Fluorosis dental endemica en Chile: estudio piloto.

The prevalence of enamel fluorosis and its severity was studied in 118 young men of 2 socio-economic levels. The subjects were born and resided for at least 6 years in Chilean communities with different natural levels of fluoride in drinking water. There was a high prevalence of enamel defect overall (54%) most of it of mild degree (36%). This was not related to the level of fluoride in drinking water, however further studies are needed since Chilean children are receiving fluoride from other sources. A national program to supplement drinking water with fluoride should take this information into account.

Study of the loosely bound non-histone chromatin proteins. Stimulation of deoxyribonucleic acid-templated ribonucleic acid synthesis by a specific deoxyribonucleic acid-binding phosphoprotein fraction.

The loosely bound chromatin proteins of Ehrlich ascites hyperdiploid cells have been prepared by extraction of chromatin with 0.35 M NaCl. Sodium dodecyl sulfate gel electrophoresis of the 0.35 M NaCl-soluble chromatin proteins reveals high heterogeneity with a molecular weight range of 10,000 to 170,000. The 0.35 M NaCl-soluble chromatin proteins contain many components similar to the more tightly bound non-histone chromatin proteins complex with the loosely bound chromatin proteins by gradient dialysis, the inhibitory effect of histones on transcription of DNA in vitro was reduced. The reconstituted complex manifested a level of template activity similar to that of native chromatin as measured in an Ehrlich ascites tumor RNA polymerase reaction. The loosely bound chromatin proteins contain RNA as well as phosphoproteins. Phenol extraction or DNA affinity chromatography of these proteins yielded fractions enhanced 25- to 30-fold in phosphorus which were capable of stimulating DNA-templated RNA synthesis in vitro. The stimulation of transcription from DNA was template-specific, effective only with a DNA template prepared from Ehrlich ascites tumor, but not from rat liver, calf thymus, or chicken erythrocytes. In addition, the stimulatory effect of the specific DNA-binding proteins appears to be RNA polymerase-specific, the stimulation being manifested with Ehrlich ascites tumor nucleoplasmic RNA polymerase and not with Micrococcus luteus RNA polymerase. Thus, the loosely bound chromosomal proteins from Ehrlich ascites tumor contain a fraction that specifically binds to Ehrlich ascites tumor DNA and exhibits a template- and RNA polymerase-specific stimulatory effect on transcription from DNA.

Characteristics of DNA-tagged liposomes allowing their use in capillary-migration, sandwich-hybridization assays.

Liposomes that have been labeled externally with a DNA oligomer are used in a capillary-migration, sandwich-hybridization assay for specific DNA target sequences. The liposomes are used in a DNA detection scheme that produces visually observable results in 10 min. The preparation and covalent attachment of a thiol-activated 22-base oligomer to the external surface of dye-containing liposomes is described, and the specificity of the assay toward perfectly complementary target DNA is demonstrated. Several characteristics of DNA-tagged liposomes that allow the use of increased stringency during hybridization are evaluated. These include the effect of temperature, formamide, and salt concentration on both the sandwich-hybridization assay and the liposomes themselves. The effects of several components of a common hybridization solution are determined with regard to both assay performance and liposome stability. Using a solution of 0.02% sodium dodecyl sulfate in 3X standard saline citrate, a visual detection limit of 200 amol of target DNA was obtained.

Evaluation of a solid-phase immunoassay for the simultaneous detection of antibodies to human immunodeficiency virus type 1 and human T-cell lymphotropic virus type I.

We describe the evaluation of a solid-phase immunoassay developed for the simultaneous detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) in human serum. The immunoassay employs a mixture of HIV-1 and HTLV-I whole viral lysates immobilized in the wells of microtiter plates. Evaluation of genetically well-pedigreed specimens along with normal blood donor samples indicated that the performance characteristics of the test were equivalent to the sensitivity and specificity of individual tests licensed by the Food and Drug Administration for antibodies to HIV-1 and HTLV-I. Furthermore, the test was also able to detect the presence of cross-reacting antibodies in HTLV-II-infected individuals. The use of such a test would greatly reduce the continually mounting costs associated with screening transfusable products for infectious agents.

Rapid method for visual identification of specific DNA sequences based on DNA-tagged liposomes.

We describe a rapid method for visually determining specific DNA sequences at femtomole concentrations. Liposomes, encapsulating a red dye and labeled with oligonucleotide, were used in a capillary migration-sandwich hybridization assay. Capture probe was immobilized on nitrocellulose strips, and liposomes, migrating along each strip, formed a visually discernible band in the presence of target DNA. One femtomole of synthetic target sequence could be detected in < 10 min. Sufficiently stringent hybridization conditions can be used to allow the discrimination of a 10% mismatch sequence from perfectly complementary DNA. A 366-base PCR product was detected at 200 fmol.

Insulin gene expression during development of the fetal bovine pancreas.

Poly(A+) RNA was isolated from the bovine pancreas at three stages of fetal development. Approximately 1% of the total RNA from first, second, and third trimester fetuses was polyadenylated, and the mean chain length of each RNA population was 1350 nucleotides. In cell-free protein synthesis experiments the concentration of insulin-immunoreactive translation products was 10.2%, 11.3%, and 9.7% for first, second, and third trimesters, respectively. Insulin mRNA sequences were estimated by transcription of insulin mRNA to [3H]cDNA and hybridization of cDNA with plasmid pI19 DNA containing rat proinsulin I sequences. Hybridization experiments gave insulin mRNA concentrations of 7.6%, 12.9%, and 3.9% for first, second, and third trimesters, respectively. These results show that insulin mRNA levels vary during development and become proportionally lower in third trimester, when the exocrine tissue is rapidly increasing in mass.

Purification of human B cell growth factor.

Human B cell growth factor (BCGF, 12,000 to 14,000 daltons) has been purified from lectin-stimulated, peripheral blood mononuclear cell-conditioned medium. The purification procedure involves a series of column chromatographic steps incorporating ion exchange, affinity binding, and gel filtration. This procedure is centered around a relatively high yield single chromatographic step, for the removal of co-eluting cytokines from BCGF, that is based on differential binding characteristics to the weak ion-exchange matrix, hydroxylapatite. Reverse-phase high-pressure liquid chromatographic separation on a C18-Bondapak column effectively separates the BCGF and TCGF moieties, yet is characterized by poor yields. High-pressure liquid chromatographic procedures on anion exchange and size exclusion provided the final purification step for BCGF, at an analytical level, resulting in a single band with a m.w. of 12,000 on a SDS-polyacrylamide gel.