Stabilization mechanism of electrodeposited silicon thin films.

Amorphous composite silicon thin films electrodeposited in tetrahydrofuran, containing up to 80 at% of Si and exhibiting an homogeneous dispersions of O, C and Cl in the amorphous Si matrix, have been successfully stabilized against oxidation using a post-annealing step in inert atmosphere. In order to understand the impact of the annealing step on their stabilization against oxidation, their composition and structure have been investigated upon heat treatments. It has been shown that the presence of impurities such as O, C and Cl does not have any impact on the stabilization process, which is rather linked to the presence of hydrogen in the Si composites. This conclusion has been drawn after a detailed analysis of the bonding structure of films annealed at different temperatures and dwell times by the mean of Raman spectroscopy. It has been shown that annealing the as-deposited films at 350 °C for a couple of hours or at higher temperatures induced a hydrogen evolution, characterized by the breaking of Si-H bonds and the formation of Si-Si bonds, which stabilized the silicon network. The understanding and the reproducibility of this stabilization process of silicon thin film electrodeposited in organic solvent paves the way for their use for many applications.

Operando analysis of a solid oxide fuel cell by environmental transmission electron microscopy.

Correlating the microstructure of an energy conversion device to its performance is often a complex exercise, notably in solid oxide fuel cell research. Solid oxide fuel cells combine multiple materials and interfaces that evolve in time due to high operating temperatures and reactive atmospheres. We demonstrate here that operando environmental transmission electron microscopy can identify structure-property links in such devices. By contacting a cathode-electrolyte-anode cell to a heating and biasing microelectromechanical system in a single-chamber configuration, a direct correlation is found between the environmental conditions (oxygen and hydrogen partial pressures, temperature), the cell open circuit voltage, and the microstructural evolution of the fuel cell, down to the atomic scale. The results shed important insights into the impact of the anode oxidation state and its morphology on the cell electrical properties.

Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle.

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum-stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.

Restoration of shp1 expression by 5-AZA-2'-deoxycytidine is associated with downregulation of JAK3/STAT3 signaling in ALK-positive anaplastic large cell lymphoma.

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALK+ ALCL) is characterized by constitutive activation of the Janus kinase (JAK)3/signal transducers and activators of transcription 3 (STAT3) signaling pathway. SHP1, a tyrosine phosphatase that negatively regulates JAK/STAT, is frequently absent in ALK+ ALCL owing to gene methylation. To test the hypothesis that loss of SHP1 contributes to JAK3/STAT3 activation in ALK+ ALCL cells, we induced SHP1 expression using 5-aza-2'-deoxycytidine (5-AZA), an inhibitor of DNA methyltransferase, in ALK+ ALCL cell lines, and correlated with changes in the JAK3/STAT3 pathway. 5-AZA gradually restored SHP1 expression in Karpas 299 and SU-DHL-1 cells over 5 days. The initially low level of SHP1 expression did not result in significant changes to the expression or tyrosine phosphorylation of JAK3 and STAT3. However, higher levels of SHP1 seen subsequently correlated with substantial decreases in JAK3 and pJAK3, followed by pSTAT3 (but not STAT3). Importantly, the decrease in JAK3 was abrogated by MG132, a proteasome inhibitor. 5-AZA induced no significant increase in apoptosis but it sensitized ALCL cells to doxorubicin-induced apoptosis. Our findings support the concept that loss of SHP1 contributes to the constitutive activation of JAK3/STAT3 in ALK+ ALCL cells. SHP1 appears to downregulate JAK3 by two mechanisms: tyrosine dephosphorylation and increased degradation via the proteasome pathway.

Role of antioxidant enzymes in the induction of increased experimental metastasis by hydroxyurea.

BACKGROUND: Treatment of tumor cells with hydroxyurea and other DNA-damaging agents has been shown to increase the experimental metastatic potential of these cells. PURPOSE: We sought to elucidate some of the biochemical and genetic changes that promote tumor cell metastasis in hydroxyurea-treated cells. We hypothesized that drug treatment induces resistance to oxidative damage and that elimination of this resistance reverses the drug-induced experimental metastatic capabilities of tumor cells. METHODS: We examined the effect of hydroxyurea treatment on B16 melanoma cells with respect to experimental metastatic potential, resistance to hydrogen peroxide (H2O2), glutathione peroxidase activity and messenger RNA (mRNA) level, glutathione reductase activity, glutathione levels, glutathione-S-transferase activity, and catalase activity and mRNA level. RESULTS: Hydroxyurea-treated cells were transiently more metastatic following intravenous injection in syngeneic mice and transiently more resistant than untreated cells to exogenous H2O2. Hydroxyurea-induced experimental metastases and H2O2 resistance were eliminated by depletion of intracellular glutathione with buthionine sulfoximine. Glutathione peroxidase activity and mRNA level, glutathione reductase activity, and reduced glutathione levels were all transiently increased in hydroxyurea-treated cells, whereas the increase in glutathione-S-transferase activity was sustained. Catalase activity was modestly increased with no increase in its mRNA levels. CONCLUSIONS: In B16 melanoma cells, experimental metastasis induced by hydroxyurea appears to depend on a process that requires glutathione. Hydroxyurea treatment also induces resistance to exogenous H2O2, which may be due to induction of glutathione and antioxidant enzyme activity. IMPLICATIONS: The role of antioxidants in B16 melanoma cells offers new insights into the metastatic process and the cellular response to chemotherapy.

Factors affecting metabolism and mutagenicity of dimethylnitrosamine and diethylnitrosamine.

For exploration of the factors affecting dimethylnitrosamine (DMN) mutagenicity, for gathering of information on the metabolism of DMN, a frequently used and relatively well-understood carcinogen, and for explanation of metabolic variations in DMN carcinogenicity, parallel in vitro assays of the microsomal activation of DMN to a mutagen and of DMN demethylation were performed. Salmonella typhimurium G46 reversions to histidine independence increase linearly with time of incubation for 30 min. At low concentrations of microsomal protein, increases in protein yield a more than proportional increase in mutations. Increasing DMN concentration saturates the enzyme, yielding less demethylation and fewer mutations proportionately. Mutagenesis is completely inhibited by 1 mM 2-diethyl-aminoethyl-2,2-diphenylvalerate. When both DMN and microsomal protein are varied at high concentrations, there is a simple linear relationship between mutagenicity and DMN demethylase activity. Thus DMN demethylase activity may be the primary controlling factor in the metabolism of DMN to a mutagen, and probably to a carcinogen; other simultaneous pathways of DMN metabolism proportional to demethylation have not been ruled out. Induction with both phenobarbital and 3-methylcholanthrene (3-MC) increased rat and mouse liver DMN demethylase activity. Mouse liver microsomes from the C57BL/6 strain demethylate DMN at a markedly lower rate than do microsomes from the C3H strain, but after 3-MC induction the relationship is reversed. Strain differences in activation of DMN were not found in the activation of diethylnitrosamine to a mutagen. Hepatic dealkylation of DMN and diethylnitrosamine to active mutagenic metabolites is increased in both rats and mice by both 3-MC and phenobarbital induction, which is in contrast to the findings of others that 3-MC and phenobarbital induction, which is in contrast to the findings of others that 3-MC decreases the incidence of DMN-induced hepatic tumors in rats, and phenobarbital decreases the incidence of diethylnitrosamine-induced hepatic tumors in mice.

A murine model of experimental metastasis to bone and bone marrow.

Bone is a common site of metastasis in human cancer. A major impediment to understanding the pathogenesis of bone metastasis has been the lack of an appropriate animal model. In this paper, we describe an animal model in which B16 melanoma cells injected in the left cardiac ventricle reproducibly colonize specific sites of the skeletal system of mice. Injection of 10(5) cells resulted in melanotic tumor colonies in most organs, including the skeletal system. Injection of 10(4) or fewer cells resulted in experimental metastasis almost entirely restricted to the skeletal system and ovary. In contrast, i.v. injection of 10(5) cells resulted in tumor colonies in the lung only. Left cardiac injection of 10(2) cells caused bone colonization, but the same number of cells injected i.v. did not colonize the lung. The number of bones with tumor colonies increased with increasing number of cells injected. Melanotic tumor colonies in the bone were characteristically distributed in the metaphysis of long bones and in the periphery of flat bones. Most animals developed paraplegia due to spinal cord compression by bony metastasis to the spine. Tumor colonization of bone occurred only in regions of bone containing hematopoietic bone marrow. This suggests that the injected tumor cells lodge, survive in the hematopoietic bone marrow environment, and grow to destroy adjacent bone. This experimental model of metastasis to bone will facilitate future studies of the pathophysiology and treatment of bone and bone marrow metastasis.

Variables affecting the killing of cultured human neuroblastoma cells with monoclonal antibody and complement.

To determine incubation conditions that result in optimal in vitro killing of human tumor cells with monoclonal antibody and complement, variables affecting the killing of cultured human neuroblastoma cells with monoclonal antibody 6-19 and baby rabbit complement were studied. Neuroblastoma target cells were stained with the fluorescent dye Hoechst 33342, which enables rapid, sensitive detection of surviving cells in conjunction with trypan blue exclusion. Maximal cell lysis was obtained at an antibody concentration of 5-10 micrograms/ml. Greater than 5 logs of cell kill were obtained with appropriate treatment conditions. No antigenic modulation was detected. Complement activity was found to be the factor which limited the extent of cell kill. Specific cell lysis increased with increasing concentration of complement. As the reaction with antibody coated cells proceeded, complement activity was depleted. This resulted in the greatest cell kill occurring during the first 10-20 min of treatment. Generation of factor(s) inhibitory to complement activity was also found. However, the effect of inhibition on limiting the extent of cell kill was much less significant than the effect of complement depletion. When compared to a single incubation of the same total duration, the reduction in complement activity with duration of incubation resulted in greater killing by multiple brief incubations with fresh complement. The depletion of complement was found to increase at a greater cell concentration. This resulted in greater proportional survival as neuroblastoma cell concentration increased. Depletion of complement activity by aggregated monoclonal antibody was found to decrease cytotoxicity. This evaluation may provide a framework for optimization of target cell destruction using complement and other monoclonal antibodies and target cells.

Fate and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in mice at a human dietary equivalent dose.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine rodent carcinogen that is found at the ppb level in cooked meat. Most laboratory studies are at 10(4)-10(7)-fold greater concentrations than actual ingested human doses. We report the first study of the bioavailability and fate of this heterocyclic amine at a human dietary equivalent dose using the high sensitivity offered by accelerator mass spectrometry. [2-14C]PhIP was administered to C57BL/6 male mice (41 ng/kg) by gavage. Tissues and excreta were collected over the subsequent 96 h. One hundred % of the administered dose was excreted in urine (90%) and feces (10%) over the length of the study. Absorption of the radiocarbon-tagged PhIP from the gastrointestinal tract was rapid, with radiocarbon levels peaking in the whole blood and urine within 1 h of exposure. Fecal 14C levels peaked at 12 h. Tissue levels peaked by 3 h with the highest concentrations of radiolabel in the intestine, stomach, and liver, followed by the kidney, pancreas, lung, and spleen. Low levels of 14C from PhIP (0.01-0.04% of the administered dose) could be detected in the tissues 48-96 h after exposure, possibly due to covalent binding to protein or DNA. The calculated half-life of PhIP at this dose was 1.14 h. This study is the first example of how accelerator mass spectrometry can be used to gather biological information about carcinogenic compounds at environmental levels of exposure.

Phosphoinositide 3-hydroxide kinase blockade enhances apoptosis in the Ewing's sarcoma family of tumors.

Ewing's sarcoma family of tumors (ESFTs) affects patients between the ages of 3 and 40 years, with most cases occurring in the second decade of life. These tumors contain a characteristic translocation, t(11;22), that produces a unique fusion protein, EWS/FLI-1. EWS/FLI-1 transforms mouse fibroblasts; this transformation requires intact EWS and FLI-1 domains as well as the insulin-like growth factor-I receptor (IGF-IR). The IGF-IR is a well-described transmembrane tyrosine kinase receptor that modulates transformation, cell growth, and survival. IGF-IR survival signaling is mediated through the downstream activation of phosphoinositide 3-OH kinase (PI 3-K) and Akt. Apoptosis, programmed cell death, progresses from a central death signal to a caspase cascade, including activation of caspase-3. Because the IGF-IR has been shown to play a role in the transformation and growth of ESFTs, we wanted to determine the role of downstream molecules in the cellular response to doxorubicin treatment. Doxorubicin increased caspase-3 activity in two ESFT cell lines, TC-32 and TC-71. Concomitant treatment of the doxorubicin-treated cells with IGF-I reduced caspase-3 activity 8-fold in TC-32 and 4-fold in TC-71. To determine whether PI 3-K has a role in IGF-I-mediated survival in ESFTs, PI 3-K was then inhibited with wortmannin and LY294002. Doxorubicin treatment reduced cell number and enhanced apoptosis in PI 3-K inhibited cells compared with noninhibited cells. Akt, a serine/threonine kinase activated downstream of PI 3-K, was investigated to determine whether its constitutive activation would render ESFT cells more resistant to doxorubicin. A constitutively activated Akt was stably transfected into ESFT and those cells with high levels of expression demonstrated increased resistance to doxorubicin-induced caspase-3 activation. These results indicate that ESFT cell lines use an IGF-IR initiated signaling pathway through PI 3-K and Akt for survival when treated with doxorubicin.

Incidence and distribution of experimental metastases in mutant mice with defective organ microenvironments (genotypes Sl/Sld and W/Wv).

Mice carrying mutations at the Sl (steel) and W (dominant white spotting) loci develop abnormalities on 3 migratory embryonic stem cell populations: hematopoietic stem cells, neural crest-derived melanocytes, and primordial germ cells. Transplantation experiments have indicated that the Sl locus affects the microenvironment where stem cells migrate, proliferate, and differentiate, while the W locus affects the migratory cells themselves. The Sl locus encodes for a multipotent growth factor known as stem cell factor. The W locus encodes the c-kit protein tyrosine kinase receptor whose ligand is the stem cell factor. We have investigated the incidence and organ distribution of experimental metastases after systemic intra-arterial injection of B16-G3.26 melanoma cells into mutant Sl/Sld and W/Wv mice. Both mutant mouse strains had a markedly lower incidence of ovarian metastases when compared with their congenic +/+ mice. In contrast to the rare colonization of the ovaries, Sl/Sld and W/Wv mice developed metastases in the myocardium, kidney, and stomach--anatomic sites that were infrequently or never affected in their congenic nonmutant mice. The only organs in which the average number of metastatic colonies differed between Sl/Sld and W/Wv mice were the bone marrow and kidneys. The average number of colonized bones per mouse in the Sl/Sld group was 5.0 +/- 3.1 (SD), compared with 12.7 +/- 5.3 in the W/Wv group. The average number of metastatic nodules in the kidneys of Sl/Sld mice was 24.6 +/- 9, while W/Wv mice had 15.5 +/- 2.5. Mutant mice with multiple metastatic nodules in the kidneys, heart, and stomach were also found to have forestomach papillomas, an enlarged duodenum, kidney abnormalities, and small body size. The results of this study provide useful information on potential mechanisms of interaction of metastatic cells with their target organs, and suggest that there are additional organ defects associated with the mutations in the Sl and W loci. They also document the importance of mutant mice in metastasis research.

In vitro and in vivo killing of acute lymphoblastic leukemia cells by L-asparaginase.

L-Asparaginase (ASNase) is a potent antileukemic enzyme routinely used in the treatment of children with acute lymphoblastic leukemia. As part of investigations of the biological activity of ASNase, we have developed techniques which measure the in vitro and in vivo cell killing ability of ASNase. To study the effect of ASNase on in vitro survival of primary lymphoblasts, bone marrow mononuclear cells obtained from untreated patients with acute lymphoblastic leukemia were cultured with and without ASNase. After 5 days, viable cells were counted using trypan blue exclusion to calculate total cell kill due to ASNase. Propidium iodide exclusion, leukemia cell surface antigens, and flow cytometry were used to determine leukemia cell kill due to ASNase. Comparison of leukemia cell kill and total cell kill showed a direct linear relationship (n = 24, r = 0.7), preferential killing of leukemia cells by ASNase (slope = 0.66), and that use of leukemia cell surface markers yielded a more accurate measurement of leukemia cell killing. ASNase at concentrations from 0.0001 to 0.1 IU/ml had equal effects on extent of leukemia cell killing (P = 0.3 to 0.7), suggesting the absence of a dose response at the ASNase concentrations tested. As a measure of the in vivo response to ASNase treatment, the number of viable bone marrow leukemia cells in the patient prior to and 5 days after treatment with ASNase was measured as the product of (% of rhodamine 123 fluorescent [viable] cells) x (absolute leukemic infiltrate). The change which occurred in the viable leukemic infiltrate was the same for patients whether they received 25,000 or 2,500 IU/m2 of ASNase as a single drug. There was a linear correlation (n = 8, r = 0.9) between in vivo and in vitro leukemia cell killing by ASNase. Thus, the in vitro assay described here can be used to predict in vivo sensitivity to ASNase in acute lymphoblastic leukemia.

Inhibition of N-myc expression and induction of apoptosis by iron chelation in human neuroblastoma cells.

Neuroblastoma is the second most common solid malignancy of childhood. Enhanced expression of the amplified N-myc gene in the tumor cells may be associated with poor patient prognosis and may contribute to tumor development and progression. The use of deferoxamine mesylate (DFO), an iron chelator, to treat neuroblastoma is being investigated in national clinical studies. We show here by TUNEL assay and DNA laddering that DFO induces apoptosis in cultured human neuroblastoma cells, which is preceded by a decrease in the expression of N-myc and the altered expression of some other oncogenes (up-regulating c-fos and down-regulating c-myb) but not housekeeping genes. The decrease in N-myc expression is iron-specific but does not result from inhibition of ribonucleotide reductase, because specific inhibition of this iron-containing enzyme by hydroxyurea does not affect N-myc protein levels. Nuclear run-on and transient reporter gene expression experiments show that the decrease in N-myc expression occurs at the level of initiation of transcription and by inhibiting N-myc promoter activity. Comparison across neuroblastoma cell lines of the amount of residual cellular N-myc protein with the extent of apoptosis measured as pan-caspase activity after 48 h of iron chelation reveals no correlation, suggesting that the decrease in N-myc expression is unlikely to mediate apoptosis. In conclusion, chelation of cellular iron by DFO may alter the expression of multiple genes affecting the malignant phenotype by multiple pathways. Given the clinical importance of N-myc overexpression in neuroblastoma malignancy, decreasing N-myc expression by DFO might be useful as an adjunct to current

Neuroblastoma: the Joint Center for Radiation Therapy/Dana-Farber Cancer Institute/Children's Hospital experience.

The treatment results for 118 patients with neuroblastoma seen at the Joint Center for Radiation Therapy/Dana-Farber Cancer Institute/Children's Hospital from 1970 to 1980 were analyzed. Patients were treated with a combination of surgery, radiation therapy, and chemotherapy depending on stage and age. Disease-free survival was excellent in all patient groups except those over one year of age with stage IV disease, a group for which currently available therapy cures only a small proportion of patients. Patients with stage III disease and older patients with stage II disease did extremely well (survival of 81% and 89%, respectively) and may have benefited from intensive treatment with all three modalities. Survival for infants (under one year) with stage IV neuroblastoma (90%) has clearly improved with intensive combination chemotherapy. With combination approaches and newer, more effective systemic regimens, a real impact on survival appears to have been made in the last decade. Better approaches will be necessary to cure more than an occasional older patient with stage IV disease.

Improved prognosis for infants with stage IV neuroblastoma.

From 1970 to 1982 11 infants with Evans stage IV neuroblastoma who were 11 months of age or less at diagnosis were treated. All but one were treated with intensive multiagent chemotherapy; eight had attempted surgical resection; only one received radiotherapy to the primary tumor. Ten of the 11 infants remain free of disease from 2 1/2 to 13 years (median, four years). Multiagent chemotherapy has clearly improved the outcome for infants with stage IV neuroblastoma.

Isochromosome 7q: the primary cytogenetic abnormality in hepatosplenic gammadelta T cell lymphoma.

Malignant lymphomas often have complex, nonrandom chromosomal abnormalities. Hepatosplenic gammadelta T cell lymphoma (gammadelta TCL) is an unusual post-thymic T cell lymphoma that primarily involves liver and spleen, often in young adult males. Few cases have had cytogenetic analysis. We report a consistent isochromosome 7q [i(7q)] abnormality in three cases of hepatosplenic gammadelta TCL, one with i(7q) as the sole abnormality at presentation. Three patients, 15-, 37- and 65-year-old males, presented with hepatosplenomegaly and fevers. Histopathologic, immunophenotypic, and molecular genetic studies supported the diagnosis. Spleen, liver, and bone marrow contained sinusoidal infiltrates of atypical lymphoid cells of T cell immunophenotype. PCR performed on two cases demonstrated clonal T cell receptor gamma gene rearrangements. Cytogenetic analysis of bone marrow showed i(7q) as the sole abnormality at presentation in one case. The second case showed i(7q) in addition to two normal chromosomes 7, and other structural and numerical abnormalities. The third case showed i(7q) and a deletion in the long arm of chromosome 11. These findings support the proposal that i(7q) represents the primary nonrandom cytogenetic abnormality in hepatosplenic gammadelta TCL, and plays a role in its pathogenesis.

Complement depletion in vitro limits monoclonal antibody 6-19-dependent complement-mediated killing of tumor cells in bone marrow.

Selective killing of nonhematopoietic tumor cells in bone marrow harvested for autologous bone marrow transplantation was studied using cultured neuroblastoma cells, monoclonal antibody 6-19, and baby rabbit serum as a source of complement. Monoclonal antibody 6-19, a murine IgG2a antibody, was selected for binding to a cell surface antigen on cultured human neuroblastoma cells that is not expressed by hematopoietic cells. The antigen is an 80-kd sialoglycoprotein present on a wide variety of nonhematopoietic tumors, and it is expressed by normal fibroblast and endothelial cells. The effect of the presence or absence of bone marrow mononuclear cells on target cell survival was evaluated using Hoechst 33342-stained neuroblastoma cells, trypan blue exclusion, and fluorescence microscopy. More than 4 logs of neuroblastoma cells were destroyed in the presence of a more than tenfold excess of bone marrow cells following two incubations with monoclonal antibody 6-19 and complement for 30 min at 37 degrees C. Monoclonal antibody concentrations greater than 5-10 micrograms/ml did not increase cell lysis. The destruction of tumor cells was limited by depletion of complement activity. Tumor cell killing increased with complement concentration and incubation duration but there was no additional cell lysis with incubations greater than 30 min. The percentage of target cells killed was significantly decreased when the target cells were treated in the presence of increasing concentrations of bone marrow mononuclear cells. This decrease in target cell destruction is a result of additional depletion of complement activity by bone marrow mononuclear cells. These results suggest that optimal purging of tumor cells by antibody and complement will be achieved following multiple brief incubations with fresh antibody and complement.

An 80,000-kd glycoprotein cell surface antigen found only on nonhematopoietic cells in human bone marrow.

The murine IgG2a monoclonal antibody (MoAb) 6-19 binds to the surface of human nonhematopoietic cells but not to hematopoietic cells. As previously described, it can be used with complement to selectively kill nonhematopoietic cells prior to culture of human bone marrow. It is now shown that the 6-19 MoAb recognizes a specific antigenic determinant of apparent molecular mass 80 kd, detected by western blotting on nonhematopoietic tumor cell lines, endothelial cells, and bone marrow stromal cells. Mouse L cells were transfected with human DNA, and the 6-19 monoclonal antibody selected cells that expressed an antigen with similar characteristics. The antigenic determinant is absent in hematopoietic tumor cell lines, peripheral blood cells, and bone marrow hematopoietic cells. Investigation of the biochemical nature of the antigen, using various enzymes, lectin-binding studies, and western blotting suggests that the 6-19 epitope is at least partially carbohydrate and that the protein has N-linked sugars but not O-linked. The affinity (Ka approximately 10(9) M-1) of MoAb 6-19 binding is similar in tumor cells and normal fibroblasts and endothelial cells. The identification of a specific antigenic determinant of 80 kd may help to discriminate between hematopoietic and nonhematopoietic cells in human bone marrow.

Grain trapping by filamentous cyanobacterial and algal mats: implications for stromatolite microfabrics through time.

Archean and Proterozoic stromatolites are sparry or fine-grained and finely laminated; coarse-grained stromatolites, such as many found in modern marine systems, do not appear until quite late in the fossil record. The cause of this textural change and its relevance to understanding the evolutionary history of stromatolites is unclear. Cyanobacteria are typically considered the dominant stromatolite builders through time, but studies demonstrating the trapping and binding abilities of cyanobacterial mats are limited. With this in mind, we conducted experiments to test the grain trapping and binding capabilities of filamentous cyanobacterial mats and trapping in larger filamentous algal mats in order to better understand grain size trends in stromatolites. Mats were cut into squares, inclined in saltwater tanks at angles from 0 to 75° (approximating the angle of lamina in typical stromatolites), and grains of various sizes (fine sand, coarse sand, and fine pebbles) were delivered to their surface. Trapping of grains by the cyanobacterial mats depended strongly on (i) how far filaments protruded from the sediment surface, (ii) grain size, and (iii) the mat's incline angle. The cyanobacterial mats were much more effective at trapping fine grains beyond the abiotic slide angle than larger grains. In addition, the cyanobacterial mats actively bound grains of all sizes over time. In contrast, the much larger algal mats trapped medium and coarse grains at all angles. Our experiments suggest that (i) the presence of detrital grains beyond the abiotic slide angle can be considered a biosignature in ancient stromatolites where biogenicity is in question, and, (ii) where coarse grains are present within stromatolite laminae at angles beyond the abiotic angle of slide (e.g., most modern marine stromatolites), typical cyanobacterial-type mats are probably not solely responsible for the construction, giving insight into the evolution of stromatolite microfabrics through time.

Microsatellite instability is infrequent in neuroblastoma.

Neuroblastoma (NB) is a childhood cancer of the autonomic nervous system. The molecular pathology of NB is not yet well understood. Both amplification of the proto-oncogene N-myc and loss of heterozygosity of several chromosomal loci occur in NB, representing genetic instability. In this study, we examined another type of genetic instability, microsatellite instability. Five chromosomal loci known to exhibit this alteration in colon, gastric, and pancreatic cancers were used in a PCR-based assay to examine 30 matched normal and tumor DNAs, which included all stages of tumor progression. Among these 30, only 2 (7%) manifested microsatellite instability. There was no correlation between the occurrence of microsatellite instability and the amplification of the N-myc gene. These data show that microsatellite instability is infrequent in neuroblastoma tumors.