Construction of a BAC contig map of chromosome 16q by two-dimensional overgo hybridization.

We have used sequence-based markers from an integrated YAC STS-content/somatic cell hybrid breakpoint physical map and radiation hybrid maps of human chromosome 16 to construct a new sequence-ready BAC map of the long arm of this chromosome. The integrated physical map was generated previously in our laboratory and contains 1150 STSs, providing a marker on average every 78 kb on the euchromatic arms of chromosome 16. The other two maps used for this effort were the radiation hybrid maps of chromosome 16 from Whitehead Institute and Stanford University. To create large sequenceable targets of this chromosome, we used a systematic approach to screen high-density BAC filters with probes generated from overlapping oligonucleotides (overgos). We first identified all available sequences in the three maps. These include sequences from genes, ESTs, STSs, and cosmid end sequences. We then used BLASTto identify 36-bp unique fragments of DNA for overgo probes. A total of 906 overgos were selected from the long arm of chromosome 16. Hybridizations occurred in three stages: (1) superpool hybridizations against the 12x coverage human BAC library (RPCI-11); (2) two-dimensional hybridizations against rearrayed positive BACs identified in the superpool hybridizations; and (3) pooled tertiary hybridizations for those overgos that had ambiguous positives remaining after the two-dimensional hybridization. For the superpool hybridizations, up to 236 overgos have been pooled in a single hybridization against the 12x BAC library. A total of 5187 positive BACs from chromosome 16q were identified as a result of five superpool hybridizations. These positive clones were rearrayed on membranes and hybridized with 161 two-dimensional subpools of overgos to determine which BAC clones were positive for individual overgos. An additional 46 tertiary hybridizations were required to resolve ambiguous overgo-BAC relationships. Thus, after a total of 212 hybridizations, we have constructed an initial probe-content BAC map of chromosome 16q consisting of 828 overgo markers and 3363 BACs providing >85% coverage of the long arm of this chromosome. The map has been confirmed by the fingerprinting data and BAC end PCR screening.

Epidemiology and clinical impact of parainfluenza virus infections in otherwise healthy infants and young children < 5 years old.

Over a 20-year period in a population of otherwise healthy children, respiratory viruses have been cultured from nasal wash specimens from each child with a clinically significant respiratory illness. Since efforts are underway to develop vaccines for prevention of illness due to parainfluenza virus (PIV) type 3, the epidemiologic characteristics of PIVs were reviewed, and the population size necessary to demonstrate vaccine efficacy was estimated. A population of 1429 children was followed through early childhood. PIVs were isolated from 286 samples, 17.4% of positive viral cultures. PIV-3 was the most common: 10% of the children had at least one symptomatic, culture-proven PIV-3 infection. PIV-3 was endemic during the study period, while the other two PIVs, PIV-1 and -2, caused biennial flu epidemics. Only four PIV-related hospitalizations were seen. The efficacy of a PIV-3 vaccine could be demonstrated in a trial of 600 carefully monitored children vaccinated by 3 months and followed to 15 months of age.

Effects of neuropeptide analogues on calcium flux and proliferation in lung cancer cell lines.

Small cell lung cancers (SCLC) and some non-small cell lung cancers (NSCLC) have neuroendocrine features which include production of a variety of neuropeptides, cell surface expression of the receptors for these peptides, and autocrine stimulation by the peptides. Previous studies showed that some peptide antagonists and anti-peptide antibodies inhibited the growth of SCLC cell lines which expressed receptors for the specific peptide. We and others showed that the heterogeneity of peptide receptor expression and responsiveness was a major potential obstacle for developing therapeutic uses of peptide antagonists. In this manuscript we evaluated the effects of 11 peptide antagonists (3 bombesin-specific, 2 cholecystokinin-specific, 1 arginine vasopressin (AVP)-specific, and 5 substance P derivatives with broad specificity) on peptide-induced calcium mobilization and growth of SCLC and NSCLC cell lines. For each antagonist, we determined the dose-response effects, specificity of peptide antagonism, and biological stability in serum using Indo-1AM-based flow cytometric assays. We found that the three bombesin antagonists, S30, SC196, and L336,175, varied in potency from 10 nM to 10 microM, varied in serum stability from 6 h to more than 24 h, and had no effect on the calcium response elicited by other peptides. None of these compounds effectively inhibited the growth of SCLC cell lines in [3H]dThd and cell growth assays in vitro. Similarly, the three cholecystokinin and AVP antagonists were highly specific for cholecystokinin and AVP, respectively, had widely varying potency, but had little inhibitory effect on SCLC growth in vitro. In contrast, the five substance P derivatives inhibited the calcium response to bombesin, AVP, bradykinin, and fetal bovine serum. None of these five antagonists were as potent as the six specific antagonists described above, but they were more effective in inhibiting the growth of SCLC cell lines in vitro. These substance P derivatives inhibited the growth of peptide-sensitive SCLC cell lines more efficiently than their inhibition of peptide-insensitive NSCLC or breast cancer cell lines. Relatively high concentrations of these substance P derivatives were required to inhibit in vitro growth, even in the absence of added peptide. It is likely that more potent broad spectrum antagonists, toxins, or radiolabeled stable antagonists will need to be developed for maximal clinical development of this type of anti-growth factor therapy.

Electromyographic analysis of exercises proposed for differential activation of medial and lateral quadriceps femoris muscle components.

BACKGROUND AND PURPOSE: The purpose of this study was to determine whether active exercises combining hip adduction with knee extension activate medial components of the quadriceps femoris muscle (QF) more than does knee extension alone. SUBJECTS: Twelve healthy adults (6 men, 6 women), aged 20 to 36 years (mean = 24.8, SD = 5.8), participated in the study. METHODS: The subjects performed quadriceps femoris setting (QS), straight leg raising (SLR), straight leg raising with the hip laterally rotated (SLR/LR), and straight leg raising combined with isometric hip adduction (SLR/ADD). Electromyographic (EMG) activity was recorded from the oblique (VMO) and longitudinal (VML) portions of the vastus medialis, vastus lateralis (VL), and rectus femoris muscles. RESULTS: Comparison of normalized mean EMG magnitudes revealed that the single-joint QF components (VMO, VML, and VL) demonstrated significantly greater activity during QS than during any of the three SLR variations and that SLR/LR and SLR/ADD did not elicit greater relative activity of medial QF components than did QS or SLR. CONCLUSION AND DISCUSSION: These findings do not support the notion that concurrent use of the hip adductors during knee extensor exercises results in preferential strengthening of the VMO.

Heterogeneity of progesterone receptor content and remodeling by tamoxifen characterize subpopulations of cultured human breast cancer cells: analysis by quantitative dual parameter flow cytometry.

Breast cancers treated with the antiestrogen tamoxifen invariably become resistant. To analyze the behavior of cell subpopulations within a tumor following tamoxifen treatment, we have used a new flow cytometry-based immunoassay and software that simultaneously quantitate PR levels and the DNA indices of ploidy and cell cycle stage in total cell populations or any subset thereof. The human breast cancer cell line T47D and its clonal derivatives were used as models of stage IV breast cancer, and growth and PR were measured as markers of antiestrogen responsiveness. We demonstrate and quantitate a remarkable heterogeneity in PR content and show the existence of distinct subpopulations with large differences in their PR levels and DNA indices, even among T47D sublines that are clonally derived. Following chronic tamoxifen treatment, an overall decrease in PR levels and growth masks an extensive heterogeneity in the response of cell subpopulations. PR levels decrease in some cells but increase in others; populations having a growth advantage expand while others contract. We find little evidence that cells, unaffected by the hormone, gain a growth advantage. Rather than exhibiting autonomy, we propose that under the influence of tamoxifen, tumors become remodeled as selected subpopulations emerge that are stimulated by the hormone, explaining the paradoxical recurrence of disease in patients undergoing endocrine therapy.

Neuropeptide signal transduction in lung cancer: clinical implications of bradykinin sensitivity and overall heterogeneity.

To define the role of neuropeptides in lung cancer biology, we evaluated the effect of seven peptide classes on signal transduction and growth in human lung and breast cancer cell lines. Flow cytometric methods were used to quantitate the calcium response in individual cells produced by these peptides alone or in combination. The effects on growth were assessed by [3H]thymidine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and soft agarose colony assays. All lung cancer cells demonstrated calcium responses to one or more peptides with classic small cell lines displaying the greatest responsiveness, followed by variant small cell lines and non-small cell lines. Breast cancer cell lines demonstrated little or no response. There was great variability in the magnitude of calcium response and pattern of response between lung cancer cell lines to individual neuropeptides. Bradykinin was the most potent peptide and produced responses in the highest fraction of lung cancer cell lines. Combinations of peptides produced greater intracellular calcium release than the single peptides, although in less than a quantitatively additive manner. Each peptide produced a refractory period which was peptide class specific. The growth stimulating effects of these neuropeptides were absent or small in magnitude and did not correlate with calcium signal transduction. These results imply that lung cancer cells display a wide sensitivity to neuropeptides but in a very heterogeneous manner. Knowledge of this heterogeneity should be incorporated into the design of antitumor strategies based on this autocrine pathway.

Effects of neuropeptides on human lung and breast cancer cells.

We evaluated the effect of seven classes of neuropeptides [bradykinin, cholecystokinin 26-33 (CCK), neurotensin, arginine-8 vasopressin (AVP), tyr-4 bombesin (BN), somatostatin, and motilin] on 18 human lung cancer and four human breast cancer cell lines to determine the pattern of responses. Flow cytometric analysis of Indo-1 AM-loaded cells was used to quantitate the intracellular calcium response of individual cells produced by these peptides alone or in simultaneous or sequential combinations. All 18 lung cancer cell lines responded to one or more peptide classes with classic small cell lines displaying the greatest responsiveness, followed by variant small-cell lines and non-small-cell lung cancer cell lines. Breast cancer cell lines demonstrated little or no response to any peptide. There was great variability in the magnitude of response and pattern of response in individual cell lines and between cell lines. Bradykinin was the most potent peptide and produced responses in the largest number of lung cancer cell lines. Simultaneous administration of active peptides produced greater intracellular calcium release than single peptides, though in a less than additive manner. Response to each peptide was followed by a refractory period lasting several hours or more. The refractoriness was peptide-specific, implying that each peptide has a distinct pathway, at least at the receptor level. Bradykinin antagonists could abrogate the calcium response to bradykinin but not to other peptides. Similarly, specific peptide antagonists for CCK, BN, and AVP blocked the response for only their specific agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Neural cell adhesion molecule expression and messenger RNA splicing patterns in lung cancer cell lines are correlated with neuroendocrine phenotype and growth morphology.

Diverse histological types of lung cancer express neuroendocrine (NE) markers. We studied the expression and alternative splicing forms of the neural cell adhesion molecule (NCAM) NKH-1, a member of the immunoglobulin superfamily, in 56 lung cancer cell lines representing all histological types. We found a strong correlation between expression of NCAM with both NE phenotype and lack of substrate adhesion in culture. Several cell lines expressed high levels of the leukocyte antigen Leu-7 (HNK-1) but were negative for NCAM antigen and mRNA, indicating that the Leu-7 antigen is distinct from NCAM. All of the NCAM-positive cell lines demonstrate a single 6.2-kilobase mRNA, and analysis of the known 3' alternative splices shows predominant expression of only the membrane form with the small intracytoplasmic domain. We conclude that (a) expression of NCAM is associated with NE phenotype regardless of the histological type of lung cancer; (b) these cell lines share a single form of NCAM; (c) with few exceptions, NCAM expression is associated with cell to cell adhesion and lack of substrate adhesion (growing as floating clusters); and (d) Leu-7 antigen is distinct from NCAM. This form of NCAM may play a functional role in NE differentiation or may be a part of the NE program expressed by these cells.

To excite a heart: a bird's view.

Ultrastructural investigations of avian cardiac muscle, including ratite hearts, have provided great insights into the mechanisms as to how excitation leads to contraction in the heart. The geometry of the conduction fibers of ratite hearts confirms earlier observations on birds showing that the geometry of the conduction system and its component cells is adapted to hearts of different sizes and rates of contraction so as to maintain a differential in conduction velocities between the conduction system and the working fibers. The study of the ratite conduction fibers bears out the idea of an inverse relationship between the size of the gap junctions and the input resistance of cardiac cells. The anomalous extended junctional SR typical of all avian hearts, proscribes the notion of direct contact transduction into calcium release for contraction of an excitatory signal propagating at the cell surface. Couplings appear well suited to maintain direct, if transitory, connections to the extracellular space in addition to harboring channels for intracellular calcium release.

Neuropeptide stimulation of calcium flux in human lung cancer cells: delineation of alternative pathways.

Calcium ion flux following the administration of a series of neuropeptides, N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, and serum was monitored by flow cytometry in selected lung and breast cancer cell lines and Chinese hamster ovary cell line CHO-K1. Calcium ion flux was monitored in individual cells by flow cytometry using the indicator indo-1 AM. Five groups of neuropeptides produced calcium flux changes in lung cancer cell lines and CHO-K1 cells but not in breast cancer cells. The peak increase in free calcium was reached within 10 sec of peptide administration and declined to resting levels in 70-120 sec. When two or more members of the same group were administered simultaneously, calcium flux changes were identical to that produced by each single peptide. When two or more members of different groups were administered simultaneously, an increased calcium release occurred. When identical peptides or peptides from the same group were administered sequentially after the return of calcium concentrations to resting values, no calcium flux resulted from the second peptide. When peptides from different active groups were administered sequentially, a new calcium flux occurred after each peptide. These data are interpreted to mean that members of each active group of peptides trigger a different calcium flux pathway. Thus, many such pathways and different metabolic states exist within the cell. Elucidation of calcium flux pathways in normal and cancer cells may lead to greater understanding of the nature of the malignant defect.

Simultaneous measurement of progesterone receptors and DNA indices by flow cytometry: characterization of an assay in breast cancer cell lines.

Progesterone receptors (PR) are the strongest predictors of response to hormone therapy in metastatic breast cancer, while PR and the DNA indices of cell ploidy and percentage of S phase are useful prognostic indicators in early stage breast cancer. We have developed a flow cytometry method to measure PR and DNA indices simultaneously using two aneuploid breast cancer cell lines--PR-positive T47D cells and PR-negative MDA-231 cells. Cells were pretreated with the progestin 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione, harvested, counted, fixed with paraformaldehyde, and permeabilized with Triton X-100. To measure total PR, cells were first exposed to a mixture of the mouse anti-PR monoclonal antibody AB-52, which binds both Protein A and Protein B of human PR, and to monoclonal antibody B-30 or B-64, which bind only Protein B. Then the cells were treated with fluorescein isothiocyanate-conjugated goat anti-mouse second antibody to produce a green fluorescence signal corresponding to PR. To measure nonspecific binding, cells were treated with mouse IgG1 as the first antibody in a parallel incubation. Specific immunoassayable PR is the difference between total and nonspecific binding. Following the antibodies, the cells were treated with RNase A and propidium iodide to give a red fluorescence signal corresponding to DNA content. Red and green fluorescence per cell was then quantified by flow cytometry. This method gives a strong specific signal for PR in several T47D cell sublines but no specific binding in MDA-231 cells. Progestin treatment led to apparent increases in PR. The proportion of cells in the G0-G1, S, and G2-M phases of the cell cycle was determined from DNA histograms and showed that both cell lines were hyperdiploid. The simultaneous flow cytometry method allowed assignment of relative PR levels in subsets of cells segregated by their DNA content. In T47D cells, PR were present throughout the cell cycle, and levels doubled in G2 and mitosis.

Automated fluorescent analysis for drug-induced cytotoxicity assays.

The human tumor clonogenic assay has been reported to predict for sensitivity of human tumors to a variety of drugs. However, this assay requires large numbers of viable cells, is time-consuming, and takes at least 2 weeks before results are available. To circumvent these problems, Weisenthal developed a microscope-based dye exclusion assay. Because this method is also time-consuming and subject to observer error, we have developed an automated method of quantitating drug cytotoxicity using a flow cytometric cell sorter (FCM). After incubation of drug-exposed tumor cells, acetaldehyde-fixed duck red blood cells (DRBC) are added. Dead tumor cells and the fixed DRBC are stained by the fluorescent dye propidium iodide, which penetrates dead cell membranes. A two-parameter analysis (cell size as measured by narrow angle light scatter vs propidium iodide fluorescence) enables determination of the live tumor cell:DRBC ratio. There was a strong correlation between the FCM method and manual counting (r = 0.958 for cell lines, r = 0.831 for fresh leukemic cells, P less than 0.0001 in both cases). We conclude that the automatized FCM method gives compatible results to the manual dye exclusion assay and increases efficiency.

Heterogeneity of cell surface antigen expression of human small cell lung cancer detected by monoclonal antibodies.

Using immunohistochemistry, radiobinding, and indirect immunofluorescence assays, seven distinct cell surface antigens, detected by monoclonal antibodies, were analyzed for the degree of homogeneity or heterogeneity of antigen expression on a panel of human small cell lung cancers. The panel included 7 tumors taken directly from patients, 21 established cell lines (9 of which were derived from different metastatic sites of 3 patients), and 33 clonal derivatives of 3 lines. With all assays, considerable heterogeneity of antigen expression between tumors from different patients was observed. In both fresh tumors and in cell lines, as well as in cell lines established from different metastatic sites in an individual patient, we observed intratumor heterogeneity finding antigen positive and negative cells and variation in antigenic density, by immunohistochemistry and indirect immunofluorescence assays. Antigenic expression was not cell cycle dependent. In addition, when cell lines or patient samples expressing antigen positive and antigen negative tumor cells were cloned, heterogeneity of antigenic expression was still present in the clonal lines. This suggests that either the expression of the antigen was not heritable and/or the ability to regenerate antigenic heterogeneity is an intrinsic property of the tumor cells. The heterogeneity of antigen expression on lung cancer cells has significant implications for the use of these and other monoclonal antibodies in the study and therapy of lung cancer.

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.

The immunological characterization of several human ribonucleases by using primary binding tests.

RNAases (ribonucleases), purified from four human tissues, as well as bovine pancreatic RNAase (RNAase A), were studied by immunodiffusion methods and by two different primary binding tests. The enzymes fell into two groups immunologically, those purified from plasma and pancreas in one and those from spleen and liver in the other. No antigenic cross-reaction between the two groups was detected by any of the immunoassays used. There was a slight antigenic cross-reaction between the human and bovine pancreatic RNAases. The liver and spleen RNAases were immunologically identical by all criteria used, whereas a small but consistent antigenic difference between the human plasma and human pancreas enzymes was detected. The significance of this difference between the human plasma and pancreas RNAases is discussed in relation to similarities and differences in their properties.

The effects of polyamines on a residue-specific human plasma ribonuclease.

A ribonuclease, purified some 2700-fold from human plasma, exhibited a strong predilection for the hydrolysis of internucleotide bonds containing cytidylic acid. Analysis of [3'-32P]- and [5'-32P]phosphoryl-terminal fragments obtained after enzymic digestion of rabbit liver and yeast RNA indicated that the nucleotide found at the 3' terminus of the fragments was invariably cytidylic acid. The nucleotide at the 5' terminus varied between cytidylic and uridylic acids in a ratio of 9:1. When characterized by DEAE-cellulose chromatography, approximately 70 per cent of the digest consisted of oligonucleotides from 4 to 8 nucleotides in length. Enzyme activity, when measured in low ionic strength buffer, could be increased severalfold above control levels by the addition of either of the polyamines, spermidine or spermine. These substances also restored nucleolytic activity to preparations inhibited by such ordered synthetic polyribonucleotides as polyguanylic acid. Estimations of the molecular weight of the enzyme, both by Sephadex gel filtration and sucrose density centrifugation, indicate that the weight may vary, depending on the presence or absence of certain cations. Of the cations examined, spermidine and spermine appear to have the greatest effect, causing an alteration in molecular weight from greater than 150,000 to approximately 32,000.