Cytogenetic characterization of olive flounder Paralichthys olivaceus: DNA content, karyotype, AgNORs and location of major ribosomal genes

A cytogenetic analysis of Paralichthys olivaceus was carried out using the flow cytometry method for DNA content, silver staining for the nucleolus organizer region （AgNORs） identification and one-color fluorescence in situ hybridization （FISH） for chromosomal mapping of major ribosomal genes. Nuclear DNA content was estimated by flow cytometry method using Gallus domesticus erythrocytes as the internal reference standard. The C-value of this species was （0.737±0.024） pg, and the DNA contents of each chromosome were estimated to be 16.51 Mb to 39.50 Mb after paired according to the average relative length. The FISH probe was made by PCR amplification of a DNA fragment containing internal transcribed spacers ITS1 between 18S and 5.8S ribosomal RNA gene, and labeled by PCR incorporation of bio-16-dUTP. FISH signals and AgNORs were both located on the secondary constrictions of chromosome 1. These results will provide a better understanding of the cytogenetic information of this species and would help for further research of the karyotype evolution in the order Pleuronectiformes.

Analysis of DNA Content of Various Types of Spermatogenic Cells in Rat after Testicular Heating with Flow Cytometry

To measure DNA contents of spermatogenic cells and analyze the efficiency of spermatogenesis after testicular heating in rat Methods Eighty adult male Sprague-Dawley rats were randomly divided into experimental group （43 ℃, 30 min） and control group （22 ℃, 30 min）. According to day 0.5, 1, 3, 6, 10, 25, 35 and 50 after local testicular heating, every group was divided into 8 subgroups： experimental subgroups （n=6） and control subgroups （n=4）. DNA contents of various types of germ cells were observed with flow cytometry （FCM） in all groups. Results Compared with control groups, percentages of 4C cell （primary spermatocyte） in 0.5-35 d groups and percentages of 1C cell （spermatid and sperm） in 6-50 d groups significantly decreased in experimental groups （P〈0.05）, and percentages of 2C cell （spermatogonium and second spermatocyte） in 3 -35 d experimental groups increased significantly after heating （P〈0.05）. 4C：2C in all of 8 experimental groups and 1C：2C in 3-35 d experimental groups were down （P〈0. 05）, and in 1 d experimental group 1C：4C was up after heating （P〈0.05）. Conclusions After being heated, the number of spermatocyte firstly decreased, and then that of spermatid and sperm decreased too. Heat influences several stages in spermatogenesis and results in suppression of spermatogenesis. Flow cytometry is an effective method for researching on the change of spermatogenesis and has significance on mechanism about changing of spermatogenic cells induced by heat.

DNA Content in Pancreatic ExocrinalCells after Vagotom yand Electron Microscopy in Rats

Fifty six SD rats were randomly divided into the normal control group and the operation group. The operating group was subdivided into six groups on the basis of killing time (the 12th h, the first day, 3rd day, first week, 2nd week and 4th week) after vagotomy (VG). The pancreatic tissues were taken for HE and Feulgen staining. The DNA content of pancreatic exocrine cells was determined by a domestically fabricated computer image analyzing system. In the control group, on the first day or in the first week after VG, the pancreatic samples were taken for transmission electron microscopic examination. The DNA content of pancreatic exocrinal cells was decreased from 1 to 3 days after VG. The secretion was found to be in inhibitory state and One week later, it gradually restored. The results indicated that the proliferation and the function of the SD rat's pancreatic exocrinal cells were prohibited at initial stage after VG, which might be concerned, at least in part, with dominance and nutrition of vagus.

EFFECTS OF PERIOPERATIVE CIMETIDINE ADMINISTRATION ON TUMOR CELL NUCLEAR MORPHOMETRY AND DNA CONTENT IN PATIENTS WITH GASTROINTESTINAL CANCER

Objective: To explore the effects of perioperative cimetidine administration on tumor cell nuclear morphometric parameters and DNA content in patients with gastrointestinal adenocarcinoma. Methods: 49 patients with pathologically confirmed gastrointestinal adenocarcinoma were randomized into test group (n=25) and control group (n=24). The test group started oral cimetidine intake 400 mg, tid, 7–10d before operation, followed by standard curative operation. The control group did not receive cimetidine. Tumor specimens were paraffin embedded for microsection and stained with hematoxylin and eosin (HE) and Feulgen stain. Morphometric studies and DNA content of tumor nuclei were performed on IBAS Image Analyzer. Results: The tumor cell nuclear area (μm2), nuclear perimeter (μm), maximal nuclear diameter (μ) for test group/control group were 23.54 5.08/34.69110.08 (P<0.001), 22.064.43/24.884.05 (P<0.05), 7.8411.64/ 8.6211.24 (P<0.05), 4.4210.61/5.4110.89 (P<0.001), Respectively. The percentages (%) of diploidy, triple-tetraploidy, quintuple ploidy, and >quintuple ploidy tumor cells for test group/control group were 16.6412.58/5.3312.14 (P<0.002), 39.8412.28/35.7013.58 (P>0.50), 12.4215.00/14.4810.74 (P>0.20), 31.1116.86/ 45.9713.82 (P<0.005), respectively. Conclusion: Perioperative administration of cimetidine in gasgtrointestinal cancer patients could decrease the nuclear size and raise the percentage of diploid tumor cells, and convert high aneuploid tumor cells into low-aneuploid tumor cells, which might help reduce the invasiveness of tumor cells.

QUANTITATIVE STUDIES ON THE DNA CONTENT AND MORPHOMETRIC FEATURES OF LIVER CELL DYSPLASIA

The DNA content and morphometric features of hepatocellular carcinoma (HCC) and liver cell dysplasia (LCD), including nuclear area, nuclear perimeter, nuclear maximum diameter and nuclear circle diameter, were quantitatively determined by means of image analysis technology. The results showed that in comparison with normal hepatocytes, LCD had a markedly increased DNA content and nuclear morphometric parameters, but the values were lower than those for HCC. LCD showed a slight increase in nuclear atypia represented by the nuclear irregular index, which was also less than HCC. The findings indicate that LCD may be a precaneerous lesion of HCC, to the cells in an abnormal proliferative state.

Flow Cytometry Analysis of Celullar DNA Content of the Parotid Pleomorphic Adenoma

The nuclear DNA content in 42 cases of primary parotid pleomorphic adenoma (PPA) and 15 cases of recurrent PPA were analysed with flowcytometer (FCM) to study the correlation of the DNA index (DI), S% and cellular proliferous index (PI) with the biologic behaivor of the tumor. The results indicated that in a portion of pleomorphic adenoma, the nuclear DNA content had altered and possessed malignant potential before any evidence of malignancy could be found under lightmicroscope. The increase of S% and PI, which followed the course of tumor development, has a close relation with the capsular invasion and the recurrence. There was a significant difference on the DI, S%, PI as well as the incidence of heteroploid between the recurrent and the incipient tumor. A great portion of recurrent tumors, which still diagnosed as benign pathologically, was acually heteroploid.

Effectof A-L Tonic Capsule on DNA Contentin Rat Experimental Hepatocarcinogenesi

The effects of A L tonic capsule on DNA content in rat experimental hepatocarcinogenesis induced by diethylnitrosamine (DENA) were observed. The experimental rats were divided into 4 groups. With exception of group D in which the rats were only administered with DENA, the rats in the groups A, B, C were previously, simultaneously and subsequently fed with A L tonic capsule respectively while they were administered with DENA. The DNA content of all rat livers was measured using automatic image analysis technique 20 weeks after administration of DENA. The results showed that the highest and lowest DNA contents were respectively seen in the groups D and A. There was significant difference in DNA contents between the groups A or B or C and D, and also between the groups A and B or C (both P <0.01). 4 components (4C) and ≥5C cells were predominant in the group D, while 2C cells were the minority. The number of 2C cells in the groups A, B, C was significantly higher than that in the group D, and the number of ≥5C cells in the groups A, B, C was markedly lower than that in the group D ( P <0.01). Also, there was very significant difference in the number of 2C and ≥5C cells between group A and B or C ( P <0.01). It was concluded that A L tonic capsule could effectively inhibit the increase of DNA content of hepatocytes and improve the distribution of DNA content in rat hepatocarcinogenesis, especially in group A.

FLOW CYTOM ETRIC ANALYSIS OF CELLULAR DNA CONTENT IN EPITHELIAL OVARIAN TUMOR

The DNA content of tumor all was analyzed by flow cytometry on parafflnembedded specimens in 73 patients with epithelial ovarian tumor, and its clinical significance was evaluated. One of the 5 benign (20%), 2 of the 11 borderline (18.18%), and 30 of the 57 malignant (52. 63%) tumors were aneuplold. The occurrence rate of aneuploidy In malignant tumors was higher than In benign and borderline tumors ( P < 0. 05 ). Furthermore, aneuploidy was more frequently In the advanced stages (Ⅲ -Ⅳ ) (77. 7%) than in the early stages (Ⅰ - Ⅱ ) (9. 5%) (P<0. 005). The occurrence rate of DNA aneuploidy was higher in patients associated with ascites and the residual tumor≥.2 cm. Patients with aneuploid tumors had more of ten ascites (P<0. 005) and residual tumor size≥2cm (P< 0.005). There was no apparent correlation between the DNA ptoidy and the histologic grade, histologic type of the tumors. G0/G1 cell proportion of DNA diplold tumors in advanced carcinoma (64. 6%) was less than those of early stage carcinoma (75. 9% ) (P<0. 05). The survival rate of diplold tumor patients was higher than that of aneuploid tumor patients in the different time after operation, and the median survival time was 30. 2 months and 10. 3 months, respectively. Multivariate analysis revealed that cellular DNA ploidy was the most Important predictive factor (P = 0. 007) of prognosis, followed by residual tumor size (P= 0. 05). Different tumor specimen of the same patient can exhibit variation sometime (38. 9%).The results revealed that the DNA ploidy may reflect tumor biological characteristics, I. e. , Its proliferative ability. Analysis of cellular DNA content of epithelial ovarian tumors would help us to predict the prognosis of the patients better.

DETERMINATION OF NUCLEAR DNA CONTENT IN LEIOMYOMA AND LEIOMYOSARCOMA OF GASTROINTESTINAL TRACT

Nuclear DNA content was measured by micro-spec trophotometry in 15 biopsy specimens from patients with gastrointestinal (GI) tract smooth muscle tumors (3 leiomyomas and 12 leiomyosarco-mas subdivided into 3 groups with 4 cases to each). The mean DNA value increased steadily as follows: leiomyoma (14.39±0.62 Au); leiomyosarcoma Grade Ⅰ (19.78±2.39 Au); leiomyosarcoma Grade Ⅱ (26.39± 1.60 Au); leiomyosarcoma Grade Ⅲ (30.66±2.39 Au). The difference of DNA value in the 4 groups had statistical significance (p<0.05-0.01). These results suggest that microspectrophotometric measurement of nuclear DNA content may serve as an objective quantitative method for the diagnosis of GI tract smooth muscle tumors and classification of leiomyosarcoma.

Evolutionary importance of the relationship between cytogeography and climate:New insights on creosote bushes from North and South America

Relationships between genome size and environmental variables suggest that DNA content might be adaptive and of evolutionary importance in plants.The genus Larrea provides an interesting system to test this hypothesis,since it shows both intra-and interspecific variation in genome size.Larrea has an amphitropical distribution in North and South American deserts,where it is most speciose.Larrea tridentata in North America shows a gradient of increasing autopolyploidy;while three of the four studied South American species are diploids,Larrea divaricata,Larrea nitida,Larrea ameghinoi,and the fourth is an allopolyploid,Larrea cuneifolia.We downloaded available focal species’georeferenced records from seven data reservoirs.We used these records to extract biologically relevant environmental variables from WorldClim at 30 arc seconds scale,to have a broad characterization of the variable climatic conditions of both regions,and a climatic envelope for each species.We estimated relative DNA content index and relative monoploid genome values,by flow cytometry,of four most abundant Larrea species throughout their respective ranges.Then we winnow the bioclimatic dataset down to uncorrelated variables and sampled locales,to analyse the degree of association between both intra-and interspecific relative DNA content and climatic variables that are functionally relevant in arid environments using Pearson correlations,general linear and mixed effects models.Within the genus Larrea,relative DNA content increases with rising temperature and decreases with rising precipitation.At the intraspecific level,all four species show relative DNA content variation across climatic conditions.Larrea is a genus that shows genome size variation correlated with climate.Our results are also consistent with the hypothesis that extreme environmental pressures may have facilitated repeated whole genome duplication events in North America,while in South America,reticulate evolution,as allopolyploidization,and speciation might have been climate-dependent since the Oligocene.

Cytotaxonomy and Cytogeography of Vicia sect. Vicilla in Northeast of China

Vicia amirensis has pollen grain with swollen mesocipium, terete style, abaxially sprouting stylar hairs and globes stigma, differing from other five species,V. amoena, V. japonica, V. pseudo-orbus, V. ramuliflora, V. unijuga andV. venosa, in sect.Vicilla of Kupicha’s System. This result further support taxonomic treatmentV. amurensis proposed by Endo and Ohash. The chromosome numbers of 64 populations and karyotypes of 15 populations for sect.Vicilla and sect.Amurense were observed.V. amurensis andV. psendo-orbus have similar karyotype features such as the same karyotype symmetry, comparable chromosome size and the same type, of Sat-chromosomes, and thereforeV. amurensis in seet.Amurense is assumed to be closely related toV. japonica in sect.Vicilla. The primary study of nuclear DNA amount was carried out on nine populations of six taxa in sect.Vicilla in China. It is primarily discussed that evolutionary trend of karyotype, nuclear DNA amount in sect.Vicilla and geographic distribution of different ploidy cytotypes.

CHANGES OF CELLULAR MORPHOLOGY AND NUCLEAR DNA CONTENT IN DIFFERENT NASOPHARYNGEAL EPITHELIUM FROM DIFFERENT PATIENTS

Cellular morphology and nuclear DNA content were measured with point-counting technique and flow cytometry (FCM) respectively in normal nasopharyngeal epithelium (NE), tissue adjacent to carcinoma (ATC) and nasopharyngeal carcinoma (NPC). The results showed that all 20 patients with NE show ed diploid, whereas 38 (38%) of 101 patients with NPC, 7 (58%) of 12 patients with recurrent NPC, 6 (30%) of 20 patients with ATC and 2 (33%) of 6 patients with NE positive to EBVCA-IgA test showed nondiploid. A total of 19 morphometric parameters of the cell were measured and calculated. The results showed that with malignant transformation of NE, most of the cellular morphometric parameters gradually increased in numerical values while no marked difference was shown between normal NE and simple hyperplasia or metaplasia in terms of cytomorphometric characteristics. Most of the parameters in dysplasia changed significantly as compared with those normal NE, simple hyperplasia or metaplasia and NPC groups. Cytomorphome

Effects ofm perioperative cimetidine administration on tumor cell nuclear morphology and DNA content in patients with gastric and colorectal cancer

Objective To explore the effects of perioperative cimetidine administration on tumor cell nuclear morphometric parameters and DNA content in patients with gastric and colorectal adenocarcinoma (GCRA) Past studies have attributed the antitumor effect of cimetidine to its immunomodulatory property, which led to an increase of cellular immunity Whether there are other possible mechanisms by which cimetidine exerts its antitumor function is unknown 49 patients with GCRA were randomized into treatment group (n=25) and control group (n=24) based on whether cimetidine was applied to them during the perioperative periold The treatment group started oral cimetidine intake 400mg, tid, 7-10d before oiperation, followed by curative surgery 'The control group did not receive cimetidine Tumor specimens were paraffin embedded for 4μm thick microsection and stained with (1) hematoxylin and eosin (HE) for the morphometric measurements of tumor cell nuclear area (NA), nuclear perimeter (NP), maximal nuclear diameter (MMND) and minimal nuclea4r diameter (MNND); (2) feulgen stain for tumor nuclear DNA content analysis by IBAS Image Analyzer The percentages (%) of diploidy (2C), tripletetraploidy (3C 4C), quintuple ploidy (5C) and >quintuple ploidy (>5C) tumor cells were calculated, using the mean value of DNA content of 50 lymphocytes as normal 2C control 3C 5C cells were designated as law aneuploid cells and >5C cells as high aneupoid cells Results The clinicopathological variables between the two groups were balanced and comparable There were no statistically significant differences between bthe treatment and control groups in regard of the following parameters: age, gender, tumor location, pathological type, TNM stage, and degree of differentiation The NA (μm 2), NP (μm), MMND (μm) and MNND (μm) for treatm ent group/control group were 23 54±5 08/34 698±10 18 ( P <0 001), 22 06±4 43/24 88±4 05 ( P <0 05),7 84±1 64/8 62±1 24 ( P >0 05), and 4 42±0 61/5 41±0 89 ( P <0 001), respectively The percentages (%) of 2C, 3C 4C, 5C and >5C tumor cells for treatment group/control group were 16 64±2 58/5 35±2 14 ( P <0 002), 39 84±2 28/35 70±3 58 ( P >0 50), 12 42±5 00/14 48±0 74 ( P >0 20), 31 11±6 86/45 97±3 82 ( P <0 005), respectively In the treatment group, there was a tendency tiowards low aneuploid tumor cells from high an euploid tumor cells However, high aneuploid tumor cells predominated in the control group Conclusion Perioperative administration of cimetidine to GCRA patients could decrease the size of tumor cell nuclei, raise the percentage of diploid tumor cells, and partially convery high aneuploid tumor cells into low aneuploid tumor cells All of these effects may in turn help reduce the proliferative potential and invasiveness of tumor cells The direct inhibitory functions on tumor cell nuclei may be a new antitumor mechanism of cimetidine, in addition to its immunomodulatory action

DNA CONTENT AND MORPHOLOGICAL PARAMETERS IN SYNOVIAL SARCOMA AND SYNOVIOMA

By using image analysis technique with 6 normal synovial tissue specimens as controls, nuclear DNA content and morphological parameters in specimens from 51 synovial sarcomas and 15 synovioma were examined quantitatively, The DNA content in synovial sarcoma and synovioma was significantly different (P < 0.01), and that of synovioma and normal synovial tissue was also different (P < 0.05), Morphological parameters including nuclear areas and perimeters differed significantly among the above three groups (P < 0.01). There was no correlation between the DNA content of synovial sarcoma and its histologic type (P > 0.05), but the DNA content greatly differed between its higher and lower differentiated grade in each type (P < 0.01). The DNA content in synovial sarcoma IIA stage, according to Enneking's staging system, was lower than that in IIB, IIIA and IIIB (P < 0.05), The 5-year survival rate of synovial sarcoma with diploid / nearly diploid (D / ND) pattern was higher than that of aneuploid (AN) pattern (P = 0.028), The study suggests that analysis of the DNA content and morphological parameters of tumor cells is helpful in the diagnosis and pathologic grading of synovial sarcoma, It also provides a guide to clinical operation and estimation of the prognosis, Although synovioma is usually a well-differentiated and benign lesion, this investigation also suggested that increased DNA content may be an indication of poor prognosis for this neoplasm.

Estimation of Nuclear DNA Content in Tannin-rich Medicinal Plant Cornus officinalis by Flow Cytometry

Objective The amount of nuclear DNA（C-value）is a key biodiversity character that provides strong unifying elements in revealing the phylogenetic regularity and relationship between genome size and functional traits for plant resource.The estimation of C-values could primarily extend our knowledge on the genetic background and genome diversity for medicinal plants,and thereby the variation of pharmacological constituents and phylogenetic mechanism of medicinal plant taxa will be revealed.However,a large number of medicinal plants（e.g.Cornus officinalis）typically contain a series of secondary metabolites,especially tannic acid,which would significantly affect the estimation of DNA content by flow cytometry（FCM）.Methodological discussions and improvement need to be made to solve this problem.Methods Two isolation buffers LB01 and Otto 1 were selected to prepare nuclear suspension with additional treatments of pre-soaking and centrifugation combination of gradient centrifugal force and duration.The best isolation and estimation methods were determined by FCM measurement in C.officinalis.Results The dry leaves were pre-soaked in Otto I buffer for 15 min and the Otto I nuclear suspension was centrifugated at 1.0×103 g for 2 min.The results showed that debris and nuclei were better separated and the scatterplots of good quality were obtained with low coefficient of variation（CV）.Contrarily,the nuclear DNA content of C.officinalis could not be accurately estimated for nuclei extracted by LB01 buffer.Finally,2C-value and genome size of C.officinalis were first estimated as 5.92 pg and 2893 Mbp,respectively.Conclusion The new methods proposed here are able to accurately estimate DNA content of C.officinalis,which provides valuable references for the estimation of genome size in other tannin-rich medicinal plants.

Molecular insights into the transgenerational inheritance of stress memory

There is accumulating evidence to show that environmental stressors can regulate a variety of phenotypes in descendants through germline-mediated epigenetic inheritance. Studies of model organisms exposed to environmental cues(e.g., diet, heat stress, toxins) indicate that altered DNA methylations, histone modifications, or non-coding RNAs in the germ cells are responsible for the transgenerational effects. In addition,it has also become evident that maternal provision could provide a mechanism for the transgenerational inheritance of stress adaptations that result from ancestral environmental cues. However, how the signal of environmentally-induced stress response transmits from the soma to the germline, which may influence offspring fitness, remains largely elusive. Small RNAs could serve as signaling molecules that transmit between tissues and even across generations. Furthermore, a recent study revealed that neuronal mitochondrial perturbations induce a transgenerational induction of the mitochondrial unfolded protein response mediated by a Wnt-dependent increase in mitochondrial DNA levels. Here, we review recent work on the molecular mechanism by which parental experience can affect future generations and the importance of soma-to-germline signaling for transgenerational inheritance.