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Proliferation of macrophages in inflammation.

Proliferation of macrophages in chronic inflammatory loci is an essential part of granuloma development and as such helps to defend the host against dissemination of harmful microorganisms. Its induction seems to depend in part on local appearance of soluble mitogenic factors since sterile cell-free exudates will induce mitotic activity in vitro in a macrophage population otherwise unable to divide. Macrophage proliferation is probably checked and controlled by inherent restrictions on the number of divisions possible. This in turn probably depends on the rapid appearance of gross chromosomal defects, the effete cells being eliminated by their incorporation into multinucleate giant cells which then form non-viable polyploid cells.

Animals↗

High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos.

For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.

Actins↗

The significance of elevated progesterone at the time of administration of human chorionic gonadotropin may be related to luteal support.

PURPOSE: Our purpose was to determine if the progesterone concentration on the day of HCG administration is associated with the establishment of pregnancy in IVF patients. METHODS: Concentrations of progesterone on the day of hCG were examined retrospectively in 293 patients with ovarian hyperstimulation induced by GnRH agonist with hMG and/or FSH. Patients were grouped based on progesterone concentration: < 0.9, 0.9 to < 1.2, 1.2 to < 1.5, and > or = 1.5 ng/ml. Oocytes recovered, fertilization rate, and pregnancy rate were compared among groups. RESULTS: Patients with a higher concentration of progesterone had increased concentrations of estradiol and greater numbers of oocytes retrieved. No differences were found for fertilization rate, polyploid fertilization, or pregnancy rate. It was noted that there was an association between the type of luteal support and the effect of high preovulatory progesterone on pregnancy rate. CONCLUSIONS: These data suggest that oocytes and embryos derived from patients with high preovulatory progesterone are not of a reduced quality. The association of high progesterone concentrations with a reduced rate of pregnancy varied with the type of luteal support.

Biomarkers↗

Efficacy of calcium ionophore A23187 oocyte activation for generating parthenotes for human embryo research.

PURPOSE: Our purpose was to examine the efficacy of Ca-A23187 to activate human oocytes and produce parthenotes for research purposes. We examined the feasibility of using fluorescence in situ hybridization (FISH) to study the sex chromosome constitution of activated oocytes. METHODS: One hundred eight nonfertilized oocytes from our IVF program were exposed to Ca-A23187. Oocyte activation was determined by the presence of pronuclear (PN) development. FISH was done on chromosome preparations using X and Y dual-colored probes. Polyploidic and parthenogenetically activated oocytes from our IVF program served as controls. RESULTS: Of the 108 oocytes, 59 (55%) had no PN, 38 (35%) one PN, 10 (9%) two PN, and 1 (0.9%) three PN. Fiftyseven oocytes (53%) were not recovered following spreading and no chromatin was observed on 14 slides (13%) after FISH. This contrasted with 50 of 227 (22%) and 3 of 227 (1.7%) loss rates, respectively, for controls (P < 0.0001). Eight of 49 activated oocytes underwent cleavage. FISH was performed on 37 oocytes. Of 21 zero-PN oocytes, I had no FISH signals, 15 had a single X, 4 had two X's, and I had four X's. For one-PN oocytes, two had no FISH signals, seven had one X, and three had two X's. For two-PN oocytes, two had no FISH signals and two had two X's. FISH results were consistent with a maternal origin of genetic material. CONCLUSIONS: Ca-A23187 resulted in a 45% activation rate, with 16% of oocytes progressing to cleavage before degeneration. Oocyte activation with Ca-A23187 allowed the generation of parthenotes for human embryo research. FISH was useful for evaluation of oocytes and parthenotes.

Blastomeres↗

Compositional patterns in the nuclear genome of cold-blooded vertebrates.

DNA preparations obtained from 122 species of fishes, 5 species of amphibians, and 13 species of reptiles were investigated in their compositional properties by analytical equilibrium centrifugation in CsCl density gradients. These species represented 21 orders of Osteichthyes, 3 orders of Chondrichthyes, 2 orders of amphibians, and 3 orders of reptiles. Modal buoyant densities of fish DNAs ranged from 1.696 to 1.707 g/cm3, the vast majority of values falling, however, between 1.699 and 1.704 g/cm3, which is the range covered by the DNAs of amphibians and reptiles. In all cases, DNA bands in CsCl were only weakly asymmetrical and only very rarely were accompanied by separate satellite bands (mostly on the GC-rich side). Intermolecular compositional heterogeneities were low in the vast majority of cases, and, like CsCl band asymmetries, at least partially due to cryptic or poorly resolved satellites. The present findings indicate, therefore, that DNAs from cold-blooded vertebrates are characterized by a number of common properties, namely a very wide spectrum of modal buoyant densities, low intermolecular compositional heterogeneities, low CsCl band asymmetries, and, in most cases, small amounts of satellite DNAs. In the case of fish DNAs a negative correlation was found between the GC level and the haploid size (c value) of the genome. If polyploidization is neglected, this phenomenon appears to be mainly due to the fact that increases and decreases in GC are associated with contraction and expansion phenomena, respectively, of intergenic noncoding sequences, which are GC poor relative to coding sequences.

Amphibians↗

Comparative mapping in grasses. Wheat relationships.

Conventionally, the genetics of species of the family Gramineae have been studied separately. Comparative mapping using DNA markers offers a method of combining the research efforts in each species. In this study, we developed consensus maps for members of the Triticeae tribe (Triticum aestivum, T. tauschii, and Hordeum spp.) and compared them to rice, maize and oat. The aneuploid stocks available in wheat are invaluable for comparative mapping because almost every DNA fragment can be allocated to a chromosome arm, thus preventing erroneous conclusions about probes that could not be mapped due to a lack of polymorphism between mapping parents. The orders of the markers detected by probes mapped in rice, maize and oat were conserved for 93, 92 and 94% of the length of Triticeae consensus maps, respectively. The chromosome segments duplicated within the maize genome by ancient polyploidization events were identified by homoeology of segments from two maize chromosomes to regions of one Triticeae chromosome. Homoeologous segments conserved across Triticeae species, rice, maize, and oat can be identified for each Triticeae chromosome. Putative orthologous loci for several simply inherited and quantitatively inherited traits in Gramineae species were identified.

Avena↗

Enzymatic cytochemistry, DNA ploidy and AgNOR quantitation in hepatocellular nodules of uncertain malignant potential in liver cirrhosis.

Conventional histological examination of echo-guided biopsy specimens can be inconclusive in small nodular lesions in cirrhotic livers. We investigated the diagnostic potential of cytochemical analysis of dipeptidyl-peptidase IV (DPP IV), of image analysis of nuclear DNA content, and of interphase silver-stained nucleolar organizer regions (AgNORs) in 12 cases of small (13- to 29-mm in diameter) hepatic nodules visualized in cirrhotic patients by ultrasonography. All cases underwent an echo-guided liver biopsy at the time of detection and in none of them were histological signs of malignancy found. Characterization with the above-mentioned techniques was always done at the time of histological examination. These patients underwent a mean (+/- SD) follow-up of 27.0 (+/- 11.2) months after biopsy, with repeated ultrasound (US) examinations. In the seven patients with subsequent neoplastic growth, DPP IV score was altered in five of six; the fraction of mononucleated polyploid cells was altered in six of seven; and the AgNOR quantity exceeded the cutoff value of 4 microns2 in five of five cases. Among the five lesions whose US appearance remained unchanged during the follow-up, only one abnormality (AgNORs) was found in one case. The combined cytochemical analysis of DPP IV, nuclear DNA content, and quantitative evaluation of interphase AgNORs in biopsy samples may contribute to the differential diagnosis of hepatocellular nodules of uncertain type in the cirrhotic liver.

Biopsy↗

Role of flhDC in the expression of the nuclease gene nucA, cell division and flagellar synthesis in Serratia marcescens.

We investigated in Serratia marcescens the functions of the flhDC operon, which controls motility and cell division in enteric bacteria. Included in our evaluations were investigation of cell division, flagellar synthesis and regulation of the expression of nuclease (encoded by the nucA(Sm) gene, one of the virulence factors). Interruption of the chromosomal flhDC operon in S. marcescens CH-1 resulted in aberrant cell division and loss of nuclease and flagella. Expression of nucA(Sm) and other mutated phenotypes was restored in the flhDC mutant by the induction of overexpression of flhDC in a multicopy plasmid. Multicopied flhDC also induced the formation of differentiated cells (polyploid aseptate cells with oversynthesis of peritrichous flagella) in broth culture using minimal growth medium. Expression of the flhDC operon showed positive autoregulation, and was growth phase dependent (upregulated in early log phase). In addition, flhDC expression was inhibited when the temperature increased from 30 to 37 degrees C, and when osmolarity was increased, but was not influenced by glucose catabolite repression. These results show that FlhD/FlhC is a multifunctional transcriptional activator involved in the process of cell differentiation, swarming and virulence factor expression.

Amino Acid Sequence↗

Polymorphism and concerted evolution in a tandemly repeated gene family: 5S ribosomal DNA in diploid and allopolyploid cottons.

5S RNA genes and their nontranscribed spacers are tandemly repeated in plant genomes at one or more chromosomal loci. To facilitate an understanding of the forces that govern 5S rDNA evolution, copy-number estimation and DNA sequencing were conducted for a phylogenetically well-characterized set of 16 diploid species of cotton (Gossypium) and 4 species representing allopolyploid derivatives of the diploids. Copy number varies over twentyfold in the genus, from approximately 1,000 to 20,000 copies/2C genome. When superimposed on the organismal phylogeny, these data reveal examples of both array expansion and contraction. Across species, a mean of 12% of nucleotide positions are polymorphic within individual arrays, for both gene and spacer sequences. This shows, in conjunction with phylogenetic evidence for ancestral polymorphisms that survive speciation events, that intralocus concerted evolutionary forces are relatively weak and that the rate of interrepeat homogenization is approximately equal to the rate of speciation. Evidence presented also shows that duplicated 5S rDNA arrays in allopolyploids have retained their subgenomic identity since polyploid formation, thereby indicating that interlocus concerted evolution has not been an important factor in the evolution of these arrays. A descriptive model, one which incorporates the opposing forces of mutation and homogenization within a selective framework, is outlined to account for the empirical data presented. Weak homogenizing forces allow equivalent levels of sequence polymorphism to accumulate in the 5S gene and spacer sequences, but fixation of mutations is nearly prohibited in the 5S gene. As a consequence, fixed interspecific differences are statistically underrepresented for 5S genes. This result explains the apparent paradox that despite similar levels of gene and spacer diversity, phylogenetic analysis of spacer sequences yields highly resolved trees, whereas analyses based on 5S gene sequences do not.

Base Sequence↗

Polymorphism and inheritance of gliadin components controlled by chromosome 6A of spring durum wheat.

The gliadin composition of 78 spring durum wheat varieties has been studied by one-dimensional (Al-lactate, pH 3.1) and two-dimensional (first dimension, Al-lactate, pH 3.1; second dimension, sodium dodecyl sulfate-polyacrylamide gel) electrophoresis. Analysis of hybrids has shown that all components of the alpha zone of gliadin spectra are inherited together as blocks and are, probably, coded for by a cluster of tightly linked genes located on chromosome 6A. Fourteen variants of gliadin blocks have been identified, which can be classified into five families on the basis of component composition. All families but one have analogues among chromosome 6A-controlled blocks of bread wheat. The results indicate that some of the genome A diploid genotypes that were ancestors of durum wheats were also ancestors of bread wheats and that polyploid wheats were produced by repeated allopolyploidization events, as has been suggested earlier.

Chromosome Mapping↗

[Expression of histocompatibility antigens in human pre-implantation embryos].

The expression of HLA antigens on polyploid human preimplantation embryos, obtained in an in vitro fertilization program, was investigated by indirect immunofluorescence test using a panel of well-defined monoclonal antibodies. Neither HLA class I antigens and beta 2-microglobulin, nor HLA class II molecules could be detected on blastomeres and the zona pellucida. It is supposed that the absence of HLA antigens on human preimplantation embryos may be one of the mechanisms important for the acceptance of the embryo by the immunocompetent mother.

Embryo Transfer↗

[Cell cycle and catamnesis in breast cancer. Cytophotometric studies].

Based on flow cytometry (120 cases) and scanning cytophotometry (56 cases) prognosis turns to the worst in the sequence diploid - polyploid - aneuploid tumors and with increasing S- and G2M-compartments. Principally the same correlation exists to other prognostic signs as lymphatic metastases, tumor size and histological grading.

Breast↗

Preliminary findings of chromosomal alterations and expression of cell cycle genes in head an neck tumors.

The genesis and progression of malignant tumors may be related to certain somatic mutations and the accumulation of multiple chromosomal alterations. Using four freshly resected malignant tumors, we investigated the relationship between chromosomal alteration and expression of cell cycle regulatory genes. Specimens of thyroid hyperplasia and normal thyroid tissue were also investigated. As cell cycle regulating genes, we chose the cdc2 gene that encodes the p34cdc2 protein kinase, a major kinase of the cell cycle, and the RCC1 gene that is essential for coupling between S and M phases. Three of the malignant tumors contained cells with chromosomal alterations, including one polyploid and two aneuploid. The DNA content of cells in thyroid hyperplasia was the same as in the normal gland. The amount of p34cdc2 protein was very low in cells of both normal thyroid and hyperplastic tissue, and grew very slowly as compared with malignant tumors. There was no significant relationship between the amount of RCC1 and ploidy pattern.

Adaptor Proteins, Signal Transducing↗

Characterization of a new human endometrial carcinoma (RL95-2) established in tissue culture.

A new human endometrial cell line, RL95-2, derived from a Grade 2 moderately differentiated adenosquamous carcinoma of the endometrium has been passaged successfully in cell culture for more than 2 yr. The cells are characteristically epithelioid with well-defined junctional complexes, tonofilaments, filopodialike extensions, and surface microvilli. Nuclei are large, irregular, and invaginated frequently with multiple, prominent, lamellar nucleoli. The cells have a log phase doubling time of 22 to 34 h followed by continued growth at a reduced rate with no apparent plateau phase. They exhibit a strong tendency for piling up as well as for the formation of glandlike dome structures. Karyotypically the line is trisomic 8 (47,XX,+8) and has an 8% frequency of polyploidization. Both cytoplasmic and nuclear estrogen receptors are present. Antihuman alpha-keratin characterizes the cell line as epithelial, nonstromal. The RL95-2 cell line may provide a useful in vitro system for the investigation of the endocrine regulation of endometrial neoplasia.

Adenocarcinoma↗

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate.

Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolonged cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.

Animals↗

Amino acid environment determines expression of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in embryonic rat hepatocytes.

A completely defined medium (EHM-1), which reflects the amino acid composition of fetal rat serum and contains albumin as the sole proteinaceous compound, allows the accumulation of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in the presence of dexamethasone, dibutyryl cyclic AMP, and triiodothyronine to approximately twice the level attained in a standard culture medium (RPMI 1640) supplemented with 10% fetal bovine serum (and hormones). Using the EHM-1 medium we could show that the capacity of hepatocytes to synthesize phosphoenolpyruvate carboxykinase in the presence of hormones is manifest as soon as the cells differentiate from the embryonic foregut (embryonic Day 11). Furthermore we could show that embryonic hepatocytes can become binuclear or polyploid when cultured in the presence of thyroid hormone.

Amino Acids↗

Investigation of effects of pregnant mare serum gonadotropin (PMSG) on the chromosomal complement of CD-1 mouse embryos.

PURPOSE: The objective of this study was to examine the effect of superovulatory doses of gonadotropins on the frequency of chromosomal abnormalities of mouse embryos. METHODS: Chromosome analysis of 8- to 16-cell stage mouse embryos and zygotes was performed by a cytogenetic method. RESULTS: There was no significant effect of the pregnant mare serum gonadotropin (PMSG) dose on the level of aneuploidy and structural abnormalities from 8- to 16-cell-stage embryos among superovulated groups. However, a simple dose-response relationship between the PMSG dose and the incidence of polyploidy was observed, with the level of polyploidy rising from 2.9% with 10 i.u. PMSG to 10.5% with 15 i.u. PMSG. In zygote stage, the proportion of polyploid embryos also increased as the dose increased, from 1.9% in 5 i.u. to 6.7% in 15 i.u. PMSG. It was observed that the extra chromosomal set in polyploidy embryos originated by both fertilization of a diploid oocyte and dispermy. CONCLUSIONS: These results indicate a dose-response relationship between the PMSG dose and the incidence of polyploidy in the CD-1 mouse. Both a disturbance at maturation division and an error at fertilization were the cause of polyploidy.

Aneuploidy↗