Chromatin Protamination and Catsper Expression in Spermatozoa Predict Clinical Outcomes after Assisted Reproduction Programs.

Identification of parameters predicting assisted reproductive technologies (ARTs) success is a major goal of research in reproduction. Quality of gametes is essential to achieve good quality embryos and increase the success of ARTs. We evaluated two sperm parameters, chromatin maturity and expression of the sperm specific calcium channel CATSPER, in relation to ART outcomes in 206 couples undergoing ARTs. Chromatin maturity was evaluated by Chromomycin A3 (CMA3) for protamination and Aniline Blue (AB) for histone persistence and CATSPER expression by a flow cytometric method. CMA3 positivity and CATSPER expression significantly predicted the attainment of good quality embryos with an OR of 6.6 and 14.3 respectively, whereas AB staining was correlated with fertilization rate. In the subgroup of couples with women ≤35 years, CATSPER also predicted achievement of clinical pregnancy (OR = 4.4). Including CMA3, CATSPER and other parameters affecting ART outcomes (female age, female factor and number of MII oocytes), a model that resulted able to predict good embryo quality with high accuracy was developed. CMA3 staining and CATSPER expression may be considered two applicable tools to predict ART success and useful for couple counseling. This is the first study demonstrating a role of CATSPER expression in embryo development after ARTs programs.

Molecular cytogenetics (FISH and fluorochrome banding): resolving species relationships and genome organization.

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Linked 5S and 45S rDNA sites are highly conserved through the subfamily Aurantioideae (Rutaceae).

Sites of 5S and 45S rDNA are more commonly located on different chromosomes of most angiosperms. Previous investigations have shown that in the subfamily Aurantioideae these sites may appear closely linked (adjacent sites), as in Poncirustrifoliata, or completely isolated, as in some species of Citrus. In the present work, the distribution of rDNA sites was investigated in representatives of 9 genera of Aurantioideae by FISH and CMA banding, aiming to understand the evolution of adjacent sites in the subfamily. A total of 57 rDNA sites were observed, 40 of them being adjacent to each other. All adjacent sites displayed the 45S rDNA array more terminally located. Assuming that the linked 5S-45S rDNA arrangement was the ancestral condition in Aurantioideae, the isolated rDNA sites observed in Clausena excavata,Bergera koenigii, and Fortunella obovata, as well as the complete linkage loss in Citrus maxima and C. medica indicates that unlinked sites arose independently several times in the evolution of the group. The linkage loss may be due to independent dispersion of one or both rDNA sequence families followed by deletion of the corresponding array in the adjacent site. The possible mechanisms behind these events and their occurrence in other groups are discussed.

Aureolic acids from a marine-derived Streptomyces sp. WBF16.

A marine-derived actinomycete (Streptomyces sp. WBF16) exhibiting antitumor activities was investigated. The strain was identified using morphological, biochemical and genetic techniques. 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces coelicolor A3 (2). Furthermore, a new aureolic acid (Chromomycin B, 1), along with Chromomycin A(2) (2) and Chromomycin A(3) (3) were isolated from its secondary metabolites. Their structures were determined by chemical and spectroscopic methods including 1D, 2D NMR and HRMS. Compounds 1-3 showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1 and HUVEC.

The chromomycin CmmA acetyltransferase: a membrane-bound enzyme as a tool for increasing structural diversity of the antitumour mithramycin.

Mithramycin and chromomycin A(3) are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl-CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.

DNA binding characteristics of mithramycin and chromomycin analogues obtained by combinatorial biosynthesis.

The antitumor antibiotics mithramycin A and chromomycin A(3) bind reversibly to the minor groove of G/C-rich regions in DNA in the presence of dications such as Mg(2+), and their antiproliferative activity has been associated with their ability to block the binding of certain transcription factors to gene promoters. Despite their biological activity, their use as anticancer agents is limited by severe side effects. Therefore, in our pursuit of new structurally related molecules showing both lower toxicity and higher biological activity, we have examined the binding to DNA of six analogues that we have obtained by combinatorial biosynthetic procedures in the producing organisms. All these molecules bear a variety of changes in the side chain attached to C-3 of the chromophore. The spectroscopic characterization of their binding to DNA followed by the evaluation of binding parameters and associated thermodynamics revealed differences in their binding affinity. DNA binding was entropically driven, dominated by the hydrophobic transfer of every compound from solution into the minor groove of DNA. Among the analogues, mithramycin SDK and chromomycin SDK possessed the higher DNA binding affinities.

Taxonomic study of a chromomycin-producing strain and reclassification of Streptomyces cavourensis subsp. washingtonensis as a later synonym of Streptomyces griseus.

A chromomycin-producing actinomycete, strain AP19, was isolated from a sample of faeces collected from Foping national nature reserve in China. Chemotaxonomic and morphological properties indicated that the novel isolate was a member of the genus Streptomyces. Phylogenetic analyses based on an almost complete 16S rRNA gene sequence of the strain and on the 120-nt nucleotide variable gamma-region of this molecule revealed that it was closely related to Streptomyces griseus ISP 5236(T) and Streptomyces cavourensis subsp. washingtonensis ATCC 27732(T). DNA-DNA relatedness values among these strains were above 70 %. Streptomyces cavourensis subsp. washingtonensis could be readily distinguished from Streptomyces cavourensis ATCC 14889(T) by differing BOX-PCR fingerprinting patterns, relatively low 16S rRNA gene sequence similarity and a low DNA-DNA relatedness value. It is proposed, therefore, that Streptomyces cavourensis subsp. washingtonensis is a later synonym of Streptomyces griseus.

Isolation and identification of newly isolated antagonistic Streptomyces sp. strain AP19-2 producing chromomycins.

A new antagonistic strain of actinomycete, designated AP19-2, was isolated from the feces of giant pandas inhabiting the Foping National Nature Reserve in China. Cultural characteristic studies strongly suggested that this strain is a member of the genus Streptomyces. The nucleotide sequence of the 16S rRNA gene of strain AP19-2 evidenced profound similarity (97-99%) with other Streptomyces strains. Two pure active molecules were isolated from a fermentation broth of Streptomyces sp. strain AP19-2 via extraction, concentration, silica gel G column chromatography, and HPLC. The chemical structures of the two related compounds (referred to as chromomycin A2 and chromomycin A3) were established on the basis of their Infrared spectra (IR), High Resolution Electrospray Ionization Mass Spectrometry (HR-ESI-MS), and Nuclear Magnetic Resonance (NMR) data, and by comparison with published data.

Relation between different human sperm nuclear maturity tests and in vitro fertilization.

PURPOSE: To consider the relationship between different sperm nuclear maturity tests and in vitro fertilization (IVF) rate, in order to select the most sensitive, specific, and independent factor(s) for prediction of in vitro fertilization. METHODS: Infertile couples (101) were randomly selected from IVF candidates referred to Isfahan Fertility and Infertility center. Semen samples were collected on the day of oocyte recovery. Following routine semen analysis, major portion of the semen was prepared for routine IVF insemination and the remaining was used for following sperm nuclear maturity tests: chromomycin A3 (CMA3), aniline blue, sodium dodecyl sulphate (SDS) test, and acridine orange test with or without heat shock (87 degrees C, 5 min). Sperms (200) were evaluated for each test. The results were recorded and analyzed for their correlation to fertilization rate, using correlation coefficient, logistic regression analysis, student t-test, and receiver operating characteristics (ROC) curve. RESULTS: Among these tests, aniline blue and CMA3, and semen parameters, sperm morphology, and sperm motility showed a significant correlation with fertilization rate. Using logistic regression analysis, sperm morphology and CMA3 were the only independent factors related to in vitro fertilization. ROC curves showed that among above tests, CMA3 is the most specific and sensitive for sperm nuclear maturity. CONCLUSION: Among CMA3, aniline blue, SDS test, and acridine orange, CMA3 was the most sensitive and specific test that can be used along with routine semen analysis for more precise prediction of fertilization rate.

Sequence specific recognition of ligand-DNA complexes studied by NMR.

The last few years have represented an accelerated accumulation in detailed information about ligand-DNA interactions. A collected view of literature information is essential for advancing our understanding of the principles of ligand-DNA recognition, utilizing this valuable information for construction of a modeling database, and eventually the rational design of DNA-binding ligands possessing desired properties. This review is concentrated on structure-based information on ligand-oligodeoxyribonucleotide (DON) complexes published since 1995, especially focusing on the results obtained from NMR structure elucidation. The discussions emphasize the sequence specific recognition of novel binding motifs or binding modules of ligand molecules rather than specific atomic details. A comprehensive list of DNA binding ligands are discussed in the text and are also summarized in a table. The DNA sequences that are recognized by specific ligand molecules as studied by NMR are annotated in a figure to provide a clear view of target selection. This review also briefly describes NMR methods for characterization and structure elucidation of ligand-DNA complexes.

Biospecific interaction analysis (BIA) of low-molecular weight DNA-binding drugs.

DNA-binding drugs have been reported to be able to interfere with the activity of transcription factors in a sequence-dependent manner, leading to alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases and viral infections. The analysis of the biological activity of DNA-binding drugs by footprinting, gel retardation, polymerase chain reaction, and in vitro transcription studies does not allow a real time study of binding to DNA and dissociation of the generated drugs/DNA complexes. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). In this study, we demonstrate that molecular interactions between DNA-binding drugs (chromomycin, mithramycin, distamycin, and MEN 10567) and biotinylated target DNA probes immobilized on sensor chips is detectable by SPR technology using a commercially available biosensor. The target DNA sequences were synthetic oligonucleotides mimicking the Sp1, NF-kB, and TFIID binding sites of the long terminal repeat of the human immunodeficiency type 1 virus. The results obtained demonstrate that mithramycin/DNA complexes are less stable than chromomycin/DNA complexes; distamycin binds to both NF-kB and TATA box oligonucleotides, but distamycin/(NF-kB)DNA complexes are not stable; the distamycin analog MEN 10567 binds to the NF-kB mer and the generated drug/DNA complexes are stable. The experimental approach described in this study allows fast analysis of molecular interactions between DNA-binding drugs and selected target DNA sequences. Therefore, this method could be used to identify new drugs exhibiting differential binding activities to selected regions of viral and eukaryotic gene promoters.

A high-resolution structure of a DNA-chromomycin-Co(II) complex determined from pseudocontact shifts in nuclear magnetic resonance.

BACKGROUND: The drug chromomycin-A(3) binds to the minor groove of DNA and requires a divalent metal ion for complex formation. (1)H, (31)P and (13)C pseudocontact shifts occurring in the presence of a tightly bound divalent cobalt ion in the complex between d(TTGGCCAA)(2) and chromomycin-A(3) have been used to determine the structure of the complex. The accuracy of the structure was verified by validation with nuclear Overhauser enhancements (NOEs) and J-coupling constants not used in the structure calculation. RESULTS: The final structure was determined to 0.7 A resolution. The structure was compared with a structure obtained in an earlier study using NOEs, in order to assess the accuracy of NOEs in giving global structural information for a DNA complex. Although some basic features of the structures agreed, they differed substantially in the fine structural details and in the DNA axis curvature generated by the drug. The distortion of base-pair planarity that was observed in the NOE structure was not seen in our structure. Differences in drug orientation and hydrogen bonding also occurred. The curvature and elongation of the DNA that was obtained previously was not found to occur in our study. CONCLUSIONS: The use of pseudocontact shifts has enabled us to obtain a high-precision global structure of the chromomycin-DNA complex, which provides an accurate template on which to consider targeting minor groove binding drugs. The effect of such binding is not propagated far along the helix but is restricted to a local kink in the axis that reverts to its original direction within four base pairs.

Mammalian artificial chromosome pilot production facility: large-scale isolation of functional satellite DNA-based artificial chromosomes.

BACKGROUND: A pilot production facility has been established to isolate mammillian artificial chromosomes at high purity by using flow cytometric techniques. Dicentric chromosomes have been generated by the targeted amplification of pericentric heterochromatic and centromeric DNA by activating the "megareplicator." Breakage of these dicentric chromosomes generates satellite DNA-based artificial chromosomes (SATAC) from 60 to 400 megabases. METHODS: For large-scale production, we have developed cell lines capable of carrying one or two SATACs. A SATAC, because of a high adenine-thymine (AT) composition, is easily identified and sorted by using chromomycin A3 and Hoechst 33258 stains and a dual laser high-speed flow cytometer. A prototype SATAC (60 megabases) has been characterized. The prototype SATAC has been isolated from an original rodent/human hybrid cell line and transferred by using modified microcell fusion into a CHO production cell line. RESULTS: Metaphase chromosomes from this production cell line were isolated in a modified polyamine buffer, stained, and sorted by using a modified sheath buffer that maintains condensed chromosomes. SATACs are routinely sorted at rates greater than 1 million per hour. Sorted SATACs have been transferred to a variety of cells by using microcell fusion technology and were found to be functional. CONCLUSIONS: By developing new SATAC containing cell lines with fewer numbers of chromosomes in conjunction with operating a high speed flow sorter we have effectively generated an efficient production facility geared purely for the isolation of SATACs.

Interaction of the DNA-binding antitumor antibiotics, chromomycin and mithramycin with erythroid spectrin.

The aureolic acid group of antitumor antibiotics, chromomycin A3 and mithramycin, are well established as transcription inhibitors, which bind reversibly to DNA at and above physiological pH, in the presence of divalent metal ions such as Mg2+. As part of our broad objective to elucidate their intracellular mode of action, other than association with DNA, we studied their interactions with the erythrocyte cytoskeletal protein, spectrin, in the absence and presence of magnesium. Different spectroscopic studies, such as absorbance, fluorescence and CD, have shown that both free chromomycin and mithramycin and their Mg2+ complexes bind to spectrin with an affinity higher than that reported for DNA. The affinity constants for the association of chromomycin and mithramycin (or their Mg2+ complexes) with spectrin are comparable with those for the association of spectrin with other cytoskeletal proteins, for example F-actin, ankyrin, protein 4.1, etc. The nature of the binding of the two antibiotics to spectrin is different. The mode of binding of the antibiotics with spectrin also changes in the presence of Mg2+. The interaction leads to a change in the tertiary structure of the protein. The relevance of the results to our understanding of the mode of action of the antibiotics is discussed.

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells.

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.

Targeting of the HIV-1 long terminal repeat with chromomycin potentiates the inhibitory effects of a triplex-forming oligonucleotide on Sp1-DNA interactions and in vitro transcription.

We have studied the effects of chromomycin and of a triple-helix-forming oligonucleotide (TFO) that recognizes Sp1 binding sites on protein-DNA interactions and HIV-1 transcription. Molecular interactions between chromomycin, the Sp1 TFO and target DNA sequences were studied by gel retardation, triplex affinity capture using streptavidin-coated magnetic beads and biosensor technology. We also determined whether chromomycin and a TFO recognizing the Sp1 binding sites of the HIV-1 long terminal repeat (LTR) inhibit the activity of restriction enzyme HaeIII, which recognizes a sequence (5'-GGCC-3') located within these Sp1 binding sites. The effects of chromomycin and the TFO on the interaction between nuclear proteins or purified Sp1 and a double-stranded oligonucleotide containing the Sp1 binding sites of the HIV-1 LTR were studied by gel retardation. The effects of both chromomycin and TFO on transcription were studied by using an HIV-1 LTR-directed in vitro transcription system. Our results indicate that low concentrations of chromomycin potentiate the effects of the Sp1 TFO in inhibiting protein-DNA interactions and HIV-1-LTR-directed transcription. In addition, low concentrations of chromomycin do not affect binding of the TFO to target DNA molecules. The results presented here support the hypothesis that both DNA binding drugs and TFOs can be considered as sequence-selective modifiers of DNA-protein interactions, possibly leading to specific alterations of biological functions. In particular, the combined use of chromomycin and TFOs recognizing Sp1 binding sites could be employed in order to abolish the biological functions of promoters (such as the HIV-1 LTR) whose activity is potentiated by interactions with the promoter-specific transcription factor Sp1.

DNA binding properties of minor groove binders and their influence on the topoisomerase II cleavage reaction.

We present titrations of the human delta beta-globin gene region with DNA minor groove binders netropsin, bisnetropsin, distamycin, chromomycin and four bis-quaternary ammonium compounds in the presence of calf thymus topoisomerase II and DNase I. With increasing ligand concentration, stimulation and inhibition of enzyme activity were detected and quantitatively evaluated. Additionally we show a second type of stimulation, the appearance of strong new topoisomerase II cleavage sites at high ligand concentrations. The specific binding sites of the minor groove binders of the DNA sequence and their microscopic binding constants were determined from DNase I footprints. A binding mechanism for minor groove binders is proposed in order to explain these results especially when ligand concentration is increased.

Targeting of the Sp1 binding sites of HIV-1 long terminal repeat with chromomycin. Disruption of nuclear factor.DNA complexes and inhibition of in vitro transcription.

Sequence selectivity of DNA-binding drugs has recently been reported in a number of studies employing footprinting and gel retardation approaches. In this paper, we studied the biochemical effects of the sequence-selective binding of chromomycin to the long terminal repeat of the human immunodeficiency type I virus. Deoxyribonuclease I (E.C.3.1.21.1) footprinting, arrested polymerase chain reaction, gel retardation and in vitro transcription experiments have demonstrated that chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1. Accordingly, interactions between nuclear proteins and Sp1 binding sites are inhibited by chromomycin, and this effect leads to a sharp inhibition of in vitro transcription.

Chronic thermotolerance with continued cell proliferation.

The human colon adenocarcinoma cell line, NSY42129, is capable of proliferation at 41.1 degrees C. This ability appears to be due to a type of chronic thermotolerance, as opposed to selection or adaptation, that allows these cells to traverse S phase at elevated temperatures. Four other human cell lines were studied for their ability to proliferate at 41.1 degrees C. Of those only one, also a colon adenocarcinoma, showed the ability to sustain proliferation at 41.1 degrees C. While all the cell lines examined showed increased levels of the major heat shock proteins at 41.1 degrees C, the cellular amounts of these proteins did not correlate with their ability to proliferate at 41.1 degrees C. However, the ability of the cells to proliferate at 41.1 degrees C did correlate with their ability to sustain elevated rates of synthesis of hsp70 and hsp90. These results could have implications in the clinical application of hyperthermia, particularly the use of long duration moderate hyperthermia.

Solution structure of mithramycin dimers bound to partially overlapping sites on DNA.

Mithramycin (MTH) is a DNA-binding antitumor agent containing A-B disaccharide and C-D-E trisaccharide segments projecting from opposite ends of an aglycone chromophore. We have previously reported on the solution structure of the MTH-DNA 6-mer complex based on a combined NMR and molecular dynamics study. This study established that the Mg(2+)-coordinated mithramycin dimer bound to a widened minor groove centered about the sequence-specific (G-C).(G-C) site and that the C-D-E trisaccharide segments from individual monomers were directed towards opposite ends of the helix spanning a six base-pair segment. This research is now extended to the binding of mithramycin dimers to partially overlapping sites on the self-complementary d(T-A-G-C-T-A-G-C-T-A) 10-mer duplex. The six base-pair mithramycin dimer footprint centered about (G-C).(G-C) steps should result in a potential steric clash in the center of the helix involving the inwardly pointing E-sugars of the pair of mithramycin dimers bound to the DNA 10-mer duplex. The MTH-d(T-A-G-C-T-A-G-C-T-A) complex (two MTH dimers per duplex) yields narrow and well-resolved NMR spectra, which have been assigned to identify intramolecular and intermolecular nuclear Overhauser enhancement (NOE) connectivities in the complex. The solution structure of the MTH-DNA 10-mer complex based on distance-restrained molecular dynamics calculations has defined the conformation of the drug and the DNA necessary for accommodation of the pair of mithramycin dimers on the DNA 10-mer helix. Specifically, the inwardly pointing E-sugars retain their face-down alignment towards the floor of the minor groove and occupy adjacent binding sites in the center of the duplex. This is achieved, in part, through torsion angle differences in the glycosidic linkage bonds along the length of the inwardly pointing aglycone-C-D-E trisaccharide segment relative to its outwardly pointing aglycone-C-D-E trisaccharide counterpart in the complex. In addition, a pronounced kink at the central (T-A).(T-A) step opens the minor groove and generates additional space to accommodate the inwardly pointing E-sugars at adjacent sites in the MTH-DNA 10-mer complex. These studies establish conformational plasticity in the C-D-E trisaccharide segment of the mithramycin dimer and deformability of the DNA helix allowing mithramycin dimers to bind to partially overlapping minor groove sites on the DNA helix.