Beyond overlap: Considering habitat preference and fitness outcomes in the umbrella species concept.

Umbrella species and other surrogate-species approaches to conservation provide an appealing framework to extend the reach of conservation efforts beyond single species. For the umbrella species concept to be effective, populations of multiple species of concern must persist in areas protected on behalf of the umbrella species. Most assessments of the concept, however, focus exclusively on geographic overlap among umbrella and background species, and not measures that affect population persistence (e.g., habitat quality or fitness). We quantified the congruence between the habitat preferences and nesting success of a high-profile umbrella species (greater sage-grouse, Centrocercus urophasianus, hereafter "sage-grouse"), and three sympatric species of declining songbirds (Brewer's sparrow Spizella breweri, sage thasher Oreoscoptes montanus, and vesper sparrow Pooecetes gramineus) in central Wyoming, USA during 2012 - 2013. We used machine-learning methods to create data-driven predictions of sage-grouse nest-site selection and nest survival probabilities by modeling field-collected sage-grouse data relative to habitat attributes. We then used field-collected songbird data to assess whether high-quality sites for songbirds aligned with those of sage-grouse. Nest sites selected by songbirds did not coincide with sage-grouse nesting preferences, with the exception that Brewer's sparrows preferred similar nest sites to sage-grouse in 2012. Moreover, the areas that produced higher rates of songbird nest survival were unrelated to those for sage-grouse. Our findings suggest that management actions at local scales that prioritize sage-grouse nesting habitat will not necessarily enhance the reproductive success of sagebrush-associated songbirds. Measures implemented to conserve sage-grouse and other purported umbrella species at broad spatial scales likely overlap the distribution of many species, however, broad-scale overlap may not translate to fine-scale conservation benefit beyond the umbrella species itself. The maintenance of microhabitat heterogeneity important for a diversity of species of concern will be critical for a more-holistic application of the umbrella species concept.

Positive ion electrospray ionization mass spectrometry of double-stranded DNA/drug complexes.

Positive ion electrospray ionization mass spectra of 16 base-pair double-stranded (ds)DNA have been obtained with essentially no ions from single-stranded DNA present. Single-stranded DNA was minimized by: (1) careful choice of DNA sequences; (2) the use of a relatively high salt concentration (0.1 M ammonium acetate, pH 8.5), and, (3) a low desolvation temperature (40 degrees C). Similarly, ESI-MS complexes of dsDNA with cisplatin, daunomycin and distamycin were obtained that contained only negligible amounts of single-stranded DNA. The complexes with daunomycin and distamycin were more stable to strand separation in the gas phase than dsDNA alone. This is in agreement with solution studies and with other recent gas phase results. These data contrast with many earlier ESI-MS studies of dsDNA and DNA/drug complexes in which ions from ssDNA are also normally observed.

Electrospray ionization mass spectrometry of oligonucleotide complexes with drugs, metals, and proteins.

I. Introduction 61 II. Binding of Small Molecules to DNA 62 A. Covalent Binding 62 B. Reversible (Noncovalent) DNA-Binding Agents 65 III. DNA-Metal Ion Complexes 67 A. Platinum Complexes 70 B. Other Metal Ions 73 IV. DNA-Protein Complexes 74 A. Introduction 74 B. ESI-MS of DNA-Protein Complexes 76 C. ESI-MS Analysis of Proteolytic Products of DNA-Protein Complexes 79 D. ESI-MS of Ternary DNA-Protein-Ligand Complexes 80 V. Conclusions 80 Abbreviations 81 References 81 --Interactions of DNA with drugs, metal ions, and proteins are important in a wide variety of biological processes. With the advent of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), mass spectrometry (MS) is now a well-established tool for the characterization of the primary structures of biopolymers. The gentle nature of the ESI process, however, means that ESI-MS is also finding application for the study of noncovalent and other fragile biomolecular complexes. We outline here the progress, to date, in the use of ESI-MS for the study of noncovalent drug-DNA and protein-DNA complexes together with strategies that can be employed to examine the binding of small molecules and metal complexes to DNA. In the case of covalent complexes with DNA, sequence information can be derived from ESI-MS used in conjunction with tandem mass spectrometry (MS/MS) and/or enzymatic digestion. MS/MS can also be used to probe the relative binding affinities of drugs that bind to DNA via noncovalent interactions. Overall, the work in this area, to date has demonstrated that ESI-MS and MS/MS will prove to be valuable complements to other structural methods, offering advantages in terms of speed, specificity, and sensitivity. (c) 2001 John Wiley & Sons, Inc.

Observation of daunomycin and nogalamycin complexes with duplex DNA using electrospray ionisation mass spectrometry.

The noncovalent binding of the antitumour drugs daunomycin and nogalamycin to duplex DNA has been studied using electrospray ionisation mass spectrometry (ESI-MS). The conditions for the preparation of drug/duplex DNA complexes and for their detection by ESI-MS have been optimised. Ions corresponding to these complexes were most abundant relative to free DNA when prepared in the pH range 8-9, and using gentle ESI interface conditions. Self-complementary oligonucleotides, 5'-d(GGCTAGCC)-3' or 5'-d(CGGCGCCG)-3', annealed in the presence of a 5-fold molar excess of either nogalamycin or daunomycin gave ESI mass spectra in which the most intense ions corresponded to three molecules of drug bound to duplex DNA, with some evidence for four drug molecules bound. For binding to 5'-d(TGAGCTAGCTCA)(2)-3', complexes containing up to four nogalamycin and six daunomycin molecules were observed. These data are consistent with the neighbour exclusion principle whereby intercalation occurs between every other base pair such that up to four bound drugs would be expected for the 8 mers and up to six for the 12 mer. Competition experiments involving a single drug in an equimolar mixture of two oligonucleotides (5'-d(TGAGCTAGCTCA)(2)-3' with either 5'-d(CGGCGCCG)(2)-3' or 5'-d(GGCTAGCC)(2)-3') showed ions arising from complexes of drug/5'-d(CGGCGCCG)(2)-3' were more intense than complexes of drug/5'-d(GGCTAGCC)(2)-3', relative to those from the 12 mer in each mixture. While this suggests ESI-MS has the potential to detect differences in sequence selectivity, more detailed experiments involving a comparison of the relative ionisation efficiency of different oligonucleotides and a wider range of intercalators are required to establish this definitively. ESI mass spectra from experiments in which both drugs were reacted with the same oligonucleotide were more complex, such that a clear preference for one drug could not be established.

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.

Numerical aberrations of chromosomes 1 and 7 in renal cell carcinomas as detected by interphase cytogenetics.

Alcohol-fixed single cell suspensions of 37 renal cell carcinomas (RCCs) were assessed by both flow cytometry (FCM) and the fluorescence in situ hybridization (FISH) technique, using chromosome 1- and chromosome 7-specific centromere DNA probes. DNA diploidy or near-diploidy was observed in 30 of the 37 RCCs and only 12 of these (near-)diploid tumours were disomic for both chromosomes 1 and 7. Numerical aberrations of chromosome 1 and/or chromosome 7 were present in 18 of the 30 (near-)diploid RCCs and five of these cases showed monosomy for chromosome 1 in more than 50 per cent of the tumour cells. A double target FISH, with a centromeric and a telomeric specific probe for 1p36, excluded misinterpretation on the basis of clustering of 1q12, and suggested a complete loss of chromosome 1. All these five (near-)diploid RCCs with monosomy for chromosome 1 were eosinophilic chromophilic cell carcinomas, according to the Thoenes classification of RCC. This observation is of special interest, because it was recently concluded from cytogenetic studies that the diagnosis of chromophilic renal cell carcinoma must be considered as obsolete. Monosomy for chromosome 1 seems to be a non-random numerical aberration of (near-)diploid eosinophilic chromophilic cell carcinomas, and a gain of one or more chromosomes 1 appeared to be a common phenomenon in RCCs, especially in the DNA aneuploid tumours. As these chromosomal abnormalities were not found in the earlier classical cytogenetic studies, we conclude that in situ hybridization techniques are required in addition to chromosome banding techniques to obtain a complete characterization of the chromosome imbalances in RCCs.

Studies on the catalytic mechanism of pig purple acid phosphatase.

Several independent experiments failed to reveal any evidence in support of the involvement of a phosphoryl-enzyme intermediate in the catalytic mechanism of pig allantoic fluid purple acid phosphatase: (i) attempts to label enzyme with phosphate derived from [32P]p-nitrophenyl phosphate were unsuccessful; (ii) values of kcat for a series of phosphate derivative varied over a wide range, with the enzyme showing a marked preference for activated ester and anhydride substrates over those with a stable leaving group; (iii) burst titrations revealed a "burst" of p-nitrophenol from p-nitrophenyl phosphate only when the enzyme was added after the substrate, suggesting that this result was an artifact of the order of addition of reagents; (iv) transphosphorylation from p-nitrophenyl phosphate to acceptor alcohols could not be detected, even under conditions where a transphosphorylation to hydrolysis ratio as low as 0.015 could have been measured; (v) enzyme-catalyzed exchange of 180 between phosphate and water was demonstrated, although at a rate much slower than that observed for other phosphatases where the involvement of a phosphoryl-enzyme intermediate in the mechanism has been clearly established. The present results are compared with those obtained in similar studies on other phosphatases, particularly the highly homologous beef spleen purple acid phosphatase, and their implications for the catalytic mechanism of the purple acid phosphatases are discussed.

DNA cytometric and interphase cytogenetic analyses of paraffin-embedded hydatidiform moles and hydropic abortions.

The combined application of DNA cytometric and interphase cytogenetic analyses was used to find objective criteria for the differential diagnosis of complete hydatidiform mole, partial hydatidiform mole and hydropic abortion. DNA ploidy and G0/G1 exceeding rates were determined using image and flow cytometric analyses on paraffin-embedded tissues of 166 cases: 71 cases of complete mole, 20 cases of partial mole, and 75 cases of abortions. To determine the existence and histological distribution of cell subpopulations with numerical chromosome aberrations, interphase cytogenetic analysis using probes specific for chromosomes 1, X, and Y was applied to paraffin tissue sections of 23 cases: 12 cases of complete mole, 3 cases of partial mole, and 8 cases of abortions. In contrast to previously reported findings that complete moles are diploid, the results of this study showed that complete moles are DNA-polyploid (96 per cent), with high G0/G1 exceeding rates and a high frequency of numerical chromosomal aberrations in the trophoblast hyperplasia. The majority of the partial moles were DNA-triploid (55 per cent). This study, however, also showed the presence of DNA-polyploid partial moles (30 per cent). Abortions were DNA-diploid (60 per cent) or DNA-triploid (39 per cent). DNA cytometric analysis, especially image DNA cytometric analysis with determination of the G0/G1 exceeding rate, and interphase cytogenetic analysis provide objective measurements which are contributory in the differential diagnosis between complete mole, partial mole, and hydropic abortion.

In situ detection of supernumerary aberrations of chromosome-specific repetitive DNA targets in interphase nuclei in human melanoma cell lines and tissue sections.

The use of non-radioactive in situ hybridization (ISH) with chromosome-specific repetitive DNA probes to study genomic changes, aneuploidy, and heterogeneity during melanocytic tumor progression, relies on its applicability to non-mitotic interphase nuclei, present in cell suspensions and tissue sections. Therefore, we studied the feasibility of detecting numerical aberrations with respect to the (peri-) centromere regions of chromosomes 1 and 7 in intact nuclei of two human melanoma cell lines with different metastatic behavior in nude mice. In addition, we used paraffin sections from xenograft lesions, obtained by inoculation of these cell lines in nude mice (subcutaneous tumors and spontaneous lung metastases). Paraffin sections from the original primary cutaneous melanoma (with a subepidermal and a dermal part) and two loco-regional metastases were also studied, one of which was the source for the cell lines. These cells and tissues represent examples of materials used in different approaches to the study of melanocytic tumor progression. Regarding the targeted sequences, ISH analysis showed that both cell lines were heterogeneous and aneuploid. The results correlated well with those obtained by ISH on metaphase spreads. Differences between the lines, which could not be detected by flow-cytometric or conventional karyotyping analysis, included data suggestive of a polyploid subpopulation and an extra copy of chromosome 7 in the metastasizing cell line. The polyploid population could be detected also in the paraffin sections of the corresponding subcutaneous xenografts and lung metastases in the mice. Both areas in the patients' primary melanoma could be evaluated separately and showed similar supernumerary aberrations of the chromosome-specific targets. These abnormalities matched those found in both metastases. Our results demonstrate that ISH can be used to visualize genomic abnormalities at the single-cell level in melanocytic nuclei in their natural context, which makes it a promising tool in the histopathology of melanocytic lesions and in the study of melanocytic tumor progression.

Establishment and characterization of five new human renal tumor xenografts.

Ten different human renal cell carcinoma (RCC) primary tumors were xenografted into BALB/c nu/nu mice. Five of the tumors (NU-10, NU-12, NU-20, NU-22, and NU-28) gave rise to serially transplantable tumors that were further characterized. Histology, DNA index, immunohistochemical characteristics, growth rate, and clonogenic potential were followed from primary tumor to the 5th to 15th transplant passage. Only one of the tumors (NU-20) showed remarkable instability for all tested parameters in the first five transplant passages. Histology of the other tumors was essentially the same to the histology of the primary tumors, although differences between human and host-derived vessels were apparent. DNA index values in general showed a trend toward an aneuploid character of the xenografts. Immunohistochemical analyses showed a loss of intensity of staining but a concomitant rise in the fraction of positively staining cells with antibodies against cytokeratins, vimentin, tumor-associated antigens, and human leukocyte antigen (HLA) class I antigens. Human leukocyte antigen class II antigen expression showed a loss of intensity as well as a decrease in the fraction of positive cells. Tumor doubling time was lowest in transplant passage number 0, and stable growth was noticed in transplant passages 1 through 4. Clonogenic potential of four of the lines was higher for the xenografts than for the primary tumors. The authors conclude that, on xenografting, histologic characteristics of the primary tumor are essentially conserved. Progression in the first transplant passages, however, results in tumors with a more aggressive character.

Chromosome detection by in situ hybridization in cancer cell populations which were flow cytometrically sorted after immunolabeling.

We examined the feasibility of performing non-radioactive in situ hybridization (ISH) in flow cytometrically sorted (tumor) cells with a chromosome #1 specific centromere probe. The study was performed in a model system of HL60 cells mixed with different quantities of HeLa cells. These latter cells were sorted directly onto poly-l-lysine coated glass slides on the basis of their keratin content, a cytoskeletal component not present in HL60 cells. Overall morphology of the separated HeLa cells was excellent and, after the ISH procedure, the appropriate number of ISH spots was observed in more than 85% of the sorted cells. This percentage did not differ significantly in cell mixtures with different percentages of HeLa cells (down to 1%). Sorting of HeLa cells in different phases of the cell cycle, and subsequent ISH, revealed the same spot number for chromosome #1 in all cell cycle stages, including mitosis. In the latter phase of the cell cycle we did not find a duplication of the chromosome #1 centromere, not even after sorting of the mitotic cells on the basis of specific labeling with an antibody to mitotin. The early G2 mitotin negative fraction, however, showed a significant percentage of cells with a duplicate spot number, most likely representing a tetraploid cell fraction in this HeLa cell culture. The protocol that evolved from these model studies was applied to cell suspensions of malignant body cavity effusions as well as solid bladder carcinomas. In several of these cases numerical chromosome aberrations could be detected by ISH more evidently after sorting on the basis of keratin labeling.

Establishment and characterization of a human melanoma cell line (MV3) which is highly metastatic in nude mice.

To select human melanoma cells that are highly tumorigenic and metastatic in nude mice we have implanted fragments of a fresh human melanoma metastasis subcutaneously (s.c.) into a nude mouse. After 3 passages in nude mice, part of the xenograft was cultured and a new melanoma cell line, MV3, was established. After intravenous (i.v.) inoculation of 2 x 10(6) MV3 cells, 95% of the nude mice (n = 20) developed lung colonies within 6 weeks. S.c. inoculation of 2 x 10(6) MV3 cells resulted in 95% tumor take, while 90% of the mice (n = 20) showed spontaneous metastases in the lungs within 7 weeks. Histological and immunohistological features of the original tumor of the patient were largely retained in the tumors of the mice and in the cell line in vitro. As shown by Alcian blue staining, MV3 cells contain large quantities of glycosaminoglycans (GAGs) and/or proteoglycanes (PGs), both in vivo and in vitro. The cells showed a marked expression of transferrin receptor, ICAM-1, EGF-receptor, and VLA-2 integrin. As only few human melanoma cell lines are available that frequently show metastasis in nude mice, the highly metastatic MV3 cell line represents a useful tool for studying the expression and regulation of molecules on human melanoma cells involved in the process of metastasis.

Dual parameter flow cytometry for deoxyribonucleic acid and intermediate filament proteins of residual mature teratoma. All tumor cells are aneuploid.

Most testicular germ cell tumors of adults are presumably derived from polyploid carcinoma in situ. Thus, one would expect that even highly differentiated teratoma components are aneuploid and that it is unlikely to find diploid tumor cell (sub)populations. We studied 10 residual mature teratomas (RMTs) using a dual parameter flow cytometry procedure. Nuclear DNA was stained with propidium iodide and cytoplasmic intermediate filament proteins, in particular, cytokeratins, with fluorescein isothiocyanate-labeled specific monoclonal antibodies. Cells in RMTs, immunoreactive with antibodies to cytokeratins were considered to be tumor cells. These were always found to be aneuploid, in agreement with the available cytogenetic data on these tumors. The diploid cells present in RMTs were devoid of cytokeratins; therefore, these cells represent the nonmalignant normal host stromal and inflammatory cells. These results, in accordance with our earlier finding, indicate that diploid testicular germ cell tumors are extremely rare in adults, and that even the histologically benign somatic tissues in RMT after polychemotherapy are aneuploid.

Negative priming in same-different matching: further evidence for a central locus of inhibition.

Responses to recently ignored information may be slower or less accurate than responses to information not recently encountered. Such negative priming effects imply that the mechanism of selective attention operates on unattended, as well as attended, information. In the present experiment, subjects judged the second and fourth letters of five-letter strings (e.g., BABAB) as "same" or "different." Responses were slower when a target letter was identical to the distractors presented in the immediately preceding trial. This effect did not depend on which response was required on the current or preceding trial. The results suggest that ignored information is functionally disconnected from the response system as a whole, rather than from a specific response.

Application of antibodies in the flow cytometric analysis of benign and malignant cells.

The application of (monoclonal) antibodies in the dualparameter flow cytometric analysis of cells, either benign or malignant, can on the one hand facilitate the recognition of their type or degree of differentiation, and on the other hand allow the quantification of cell populations at different stages of the cell cycle or with certain cell biological characteristics. The usefulness and advantages of this approach are illustrated here on the basis of studies with antibodies to specific keratins which make up part of the epithelial cytoskeleton.

Detection of numerical chromosome aberrations in bladder cancer by in situ hybridization.

The nuclear DNA content of 53 transitional cell carcinomas (TCCs) of the urinary bladder, as determined by flow cytometry (FCM), was compared with chromosome ploidy as detected by nonradioactive in situ hybridization (ISH). For this purpose, probes for repetitive DNA targets in the (peri) centromeric region of chromosomes 1 and 18 were used. Hybridization results with both probes of 35 TCCs, which had a DNA index of approximately 1.0 as concluded from FCM, showed evident chromosome 1 aberrations in approximately 25% of the tumors, and in a few cases an aberration for chromosome 18 was detected. Comparison of the ISH spot numbers for both chromosomes showed in most cases a higher number for chromosome 1 than for chromosome 18. ISH on 18 cases of TCCs, which showed a single peak in FCM with a DNA-index of 1.2 to 3.2, exhibited a profound heterogeneity. In these TCCs the ratio between chromosomes 1 and 18 varied over a wide range, resulting in cases showing more hybridization signals for chromosome 1 than for chromosome 18 or the opposite. Furthermore, using ISH minor cell populations showing polyploidization and giant cells containing numerous ISH signals could occasionally be detected. Results showed that interphase cytogenetics by ISH enable a fast screening of numerical chromosome aberrations and detection of different cell populations within one tumor, which was apparently homogeneous according to FCM.

Image and flow DNA cytometry of small cell carcinoma of the lung.

Both image and flow DNA cytometry were performed in isolated nuclei from paraffin-embedded tumor tissue of patients with small cell carcinoma of the lung (SCCL). In 14 patients tissue was obtained by surgery from the primary tumor. From 14 patients tissue was taken by autopsy. From two patients tissue obtained by both surgery and later autopsy were available. From the autopsy patients tissue was taken only from the primary tumor (n = 6), from a metastasis (n = 1) and from the primary tumor and distant metastases (n = 7). Twelve of the tumors obtained by surgery were diploid, and two multiploid (two stem lines present). This was found both with image and flow cytometry. The group of patients could clearly be subdivided in short survivors (less than 9 months, n = 6) and long survivors (greater than 16 months, n = 8); since in both groups one multiploid and the remainder diploid cases were present, ploidy did not seem to be a good prognosticator for survival. In most (n = 26) of the tissues measured from the autopsy patients, again, a good correlation between image and flow DNA cytometry was obtained, the histograms being either (near) diploid or multiploid. In six cases, however, flow cytometry showed multiploidy whereas image showed aneuploidy (one single peak clearly deviating from diploidy). This discrepancy is caused because normal diploid (nonneoplastic) cells in the preparations could not be discarded from the flow cytometry measurements. Using the image cytometry data of the primary tumors, five diploid, three aneuploid, and four multiploid tumors were found. In five of the seven patients of whom tissue was obtained from the primary tumor and multiple metastases, differences between the histograms were found, mostly showing two malignant cell populations in one tissue and only one of them in another. Of one of the two patients of whom tissue was obtained by surgery and later autopsy, a change in histogram pattern was observed. It is concluded that although there is a high similarity between image and flow DNA cytometry, for an optimal interpretation of the histogram pattern, image measurements are more reliable. Ploidy determination does not seem to be of use in prediction of survival, and care should be taken in interpreting DNA histograms of metastases in SCCL patients because of the variability in histogram pattern.

ETN-1: a new human endometrial carcinoma cell line producing ascites and distant metastases in nude mice.

A human carcinoma cell line (ETN-1) has been established from a skin metastasis of a moderately differentiated adenocarcinoma of endometrial origin. The cell line has been so far maintained for 27 months through 55 passages, growing as a monolayer as well as in 3-dimensional clusters with a population doubling time of 72 hr. The number of chromosomes per cell varied from 39 to 107 (average number 61.0 +/- 19.8), with a modal number of 46-48. Seven clonal marker chromosomes were detected. Flow cytometric analysis revealed a population of pseudo-tetraploid cells (DNA index 2.1) next to a pseudo-diploid population (DNA index 1.1). The epithelial character of the cells was confirmed by a positive immunocytochemical reaction using monoclonal antibodies (MAbs) to different keratins, the epithelial cell markers BW 495/36 and HMFG-2, as well as by the presence of many junctional complexes. The tumour cells retained a positive reaction with the anti-ovarian carcinoma OV-TL 3, OV-TL 10 and OC 125 MAbs, although the reaction was markedly diminished in comparison with the original tumour. Tumour cells inoculated subcutaneously in nude mice produced well differentiated adenomatous tumour nodules with formation of glandular lumina and basal lamina. Tumour cells injected intraperitoneally produced malignant ascites and regional as well as distant metastases of adenomatous character.

Ki-67 detects a nuclear matrix-associated proliferation-related antigen. II. Localization in mitotic cells and association with chromosomes.

In interphase cells the proliferation-associated antigen recognized by monoclonal antibody Ki-67 is almost exclusively located in the nucleoli. When cells at several stages of mitosis were examined for the localization of the Ki-67 antigen, a striking redistribution could be observed. During prophase the distinct nucleolar Ki-67 fluorescence changed to a bright irregular meshwork throughout the nucleoplasm. At metaphase the antigen appeared to be distributed in a reticulate structure surrounding the condensed chromosomes, while at late telophase a punctated staining of the entire nucleoplasm was observed, which preceded the typical nucleolar localization pattern in each of the two daughter cells. Immunolabelling with Ki-67 of metaphase chromosome spreads revealed a circumferential staining of the individual chromosomes. The Ki-67 antigen is preserved in nuclear matrix preparations obtained after in situ fractionation of interphase cells. When mitotic cells were exposed to such treatments, the obtained fluorescence data suggested that the antigen may be part of the chromosome scaffold. Quantification of the Ki-67 fluorescence signal using flow cytometry revealed the highest staining intensities in mitotic cells. Furthermore, it was shown that nutritionally deprived cells became negative for Ki-67.

Role of sequence amplification in the generation of nematode mitochondrial DNA polymorphism.

Romanomermis culicivorax, an obligate parasitic nematode of mosquitos, possesses an unusually large mitochondrial genome. Individuals are monomorphic for one of several mitochondrial DNA (mtDNA) size variants ranging from 26-32 kb. In this report, we demonstrate that the mitochondrial genome size differential in three isofemale lineages is due to the presence of mtDNA sequences amplified to different copy numbers within each mtDNA molecule. Restriction enzyme analysis and DNA sequencing studies reveal that each mitochondrial genome contains one of two 3.0 kb repeat types that differ by approximately 30 bp. This difference is primarily due to a short (23 bp) imperfect tandem duplication present within the larger of two polymorphic repeating units. The 3.0 kb reiterated DNA sequences are present as direct, tandem repeats and as inverted portions of the same sequence located elsewhere in the genome. Based on mtDNA analysis of an independently reared R. culicivorax culture, we conclude that events resulting in mitochondrial genome rearrangement occurred in natural field populations prior to propagation within the laboratory.