[Clinical outcomes of intracytoplasmic sperm insemination with micro amount frozen-thawed diagnostic sperm in the treatment of azoospermia].

Objective: To investigate the efficacy and clinical outcomes of intracytoplasmic sperm injection (ICSI) with micro amount frozen-thawed diagnostic sperm obtained by microdissection testicular sperm extraction (microTESE), percutaneous epididymal sperm as-piration (PESA) and testicularsperm extraction (TESA) in the treatment of azoospermia. Methods: A retrospective analysis was performed on 736 ICSI cycles of azoospermia patients.In Reprocluctive Medicine Center of the First Affiliated Hospital of Zhengzhou University from January 2018 to December 2019. Including 199 ICSI cycles (microTESE 47cycles, PESA 75cycles and TESA 77 cycles) with micro amount frozen-thawed diagnostic sperm and 537 ICSI cycles (microTESE 23 cycles, PESA 111 cycles and TESA 403 cycles) with fresh micro amount sperm. The general conditions, embryo development conditions and clinical outcomes of patients were compared between and within the two groups. Results: The recovery rate of PESA group was significantly lower than that of TESA group (89.3% vs 98.7%), P<0.05. The rate of 2PN in the fresh control group was significantly higher than that in the experiment group (75.5% vs 71.3%) and the rate of 2PN in the fresh microTESE and PESA groups were also significantly higher than those of the frozen-thawed microTESE and PESA groups (74.2% vs 64.6%) and (78.5% vs 72.4%), P<0.05. Both the rate of D5 blastocyst formation and high quality blastocyst in the fresh group were significantly lower than that in the experiment group (26.9% vs 32.9%) and (15.1% vs 18.0%), P<0.05; both the rate of early cleavage and blastocyst formation in the fresh microTESE group were significantly lower than that in the frozen-thawed microTESE group (55.1% vs 68.3%) (27.3% vs 39.3%), P<0.05. Both the rate of 8 cells embryos and blastocyst formation in the fresh TESA group were significantly lower than those of the TESA frozen-thawed group (41.3% vs 46.0%) (26.5% vs 32.4%), P<0.05. There was no significant difference in pregnancy rate and planting rate between or within the groups(P>0.05). The abortion rate in the frozen-thawed group was significantly higher than the fresh group (12.0% vs 4.0%), P<0.05, especially the abortion rate in the PESA frozen-thawed group was significantly higher than the fresh group (18.0% vs 1.7%), P<0.05. There was no significant difference in gender, weight and body length between the fresh group and the frozen-thawed group (P>0.05), but there were two malformed babies born in the frozen-thawed group. Conclusions: Frozen-thawed microinjection of diagnostic microspermatozoa is a feasible method for the treatment of asthenospermia.There was on significonty difference in pregnancy rate and planting rate between of with in the groups. However, significantly higher than the fresh PESA group of the influence on offspring needs to be further studied.

[Effects of different ages and body mass index on embryo development time kinetic parameters and embryo development potential].

Objective: To investigate the effects of age and body mass index (BMI) on embryo development time kinetic parameters, embryo development potential and clinical pregnancy outcomes. Methods: A retrospective study was conducted to analyze the data of 6 294 embryos from 832 patients who underwent in vitro fertilization and embryo transfer (IVF-ET) in the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from September 2016 to November 2018. According to the age, they were divided into two groups:<35-year-old group (655 cases, 5 076 embryos), ≥35-year-old group (177 cases, 1 218 embryos). According to the BMI, they were divided into three groups: low body mass group (BMI<18.5 kg/m(2), 47 cases, 355 embryos), normal body recombination (18.5-23.9 kg/m(2), 517 cases, 3 813 embryos), hyperrecombination (BMI>23.9 kg/m(2), 268 cases, 2 126 embryos). Embryo development time kinetic parameters, embryo development potential and clinical pregnancy outcomes in each group were compared. Results: Embryo development to 3 cells, 4 cells were faster in <35-year-old group than in ≥35-year-old group. The blastocyst formation rate, high-quality blastocyst formation rate, pregnancy rate, implantation rate, delivery rate, live birth rate, and abortion rate were all statistically significant (all P<0.05). There were no significant differences in normal fertilization rate, cleavage rate, embryo utilization rate, high quality embryo rate, pregnancy rate, implantation rate, abortion rate, delivery rate, live birth rate between the three BMI groups (all P>0.05). Conclusions: The age has an effect on the partial embryo development time kinetic parameters, but BMI has a little effect on it.

[Prediction of clinical outcomes of patients with surplus cleavage-stage embryos for blastocyst culture after D3 embryo transfer].

Objective: To find the best strategy of embryo transfer, so as to provide theoretical basis for improving the clinical outcomes of in vitro fertilization-Embryo transfer (IVF-ET), we investigate the blastocyst culture of surplus cleavage-stage embryos after D3 embryo transfer and the prediction of clinical outcomes with or without blastocyst formation. Methods: A retrospective study was conducted on 3 568 patients who underwent IVF-ET in the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from January 2016 to May 2018, whotransplanted two embryos in D3 with blastocyst culture of surplus cleavage-stage embryos, according to their age, they were divided into three groups: <35 years old group, 35-38 years old group, and>38 years old group.And according to the presence or absence of blastocyst formation, they were also divided into two subgroups: blastocyst formation group and non-blastocyst formation group. The embryo development and clinical outcomes in each group were compared. Results: (1) Comparisons of the embryo development in the three age groups with the first cycle. The total fertilization rate, cleavage rate and high quality embryo rate of the blastocyst formation group in the three groups were higher than those in the non-blastocyst formation group, P<0.05; In<35 years old group, the embryo utilization rate (75.0% vs 70.6%), pregnancy rate (74.9% vs 70.3%), planting rate (53.6% vs 48.6%), delivery rate (66.7% vs 61.1%) and live birth rate (66.5% vs 61.0%) of the blastocyst formation group were higher than those in the non-blastocyst formation group, P<0.05. (2) Comparisons of embryo development in the three age groups with multiple cycles (≥2 cycles). In<35 years old group, the total fertilization rate (75.0% vs 70.6%),delivery rate (62.7% vs 43.8%) and live birth rate (62.7% vs 43.8%) of the blastocyst formation group were significantly higher than those in the non-blastocyst formation group, P<0.05; In>38 years old group, the pregnancy rate (56.3% vs 25.8%), implantation rate (34.4% vs 14.5%), delivery rate (43.8% vs 11.3%), live birth rate (43.8% vs 11.3%) of the blastocyst formation group were higher than those in the non-blastocyst formation group, P<0.05. Conclusions: The results of blastocyst culture in different groups can predict the outcomes of embryo transfer in D3. For patients<35 years old with the first cycle, the clinical outcomes of the blastocyst formation group after D3 embryo transfer is better than that of the non-blastocyst formation group. For Patients with multiple cycles (≥2 cycles),the clinical outcomes of the embryo formation group is superior to that of the non-blastocyst formation group<35 years old or>38 years old.

Group housing with young mice relieves Alzheimer's disease behaviors in aging mice.

OBJECTIVE: Due to the increase in human life expectancy, a higher number of individuals are experiencing age-related cognitive impairments. Therefore, it is important to investigate the methods to tackle the effects of aging. MATERIALS AND METHODS: The mice were divided into the following groups: the aging mice (male, 20 months) and young mice (male, 2 months) were pairing-housed together in the same cage and lasted for at least one month (Aging-Young). The following tests were performed for the mice in different groups: Open field test, Morris water maze (MWM) test, Contextual fear conditioning test, Novel Object Recognition (NOR) test, Pain threshold test, Olfactory habituation/dishabituation test, T-maze test, Electrophysiological recordings. RESULTS: In this study, we housed aging and young mice together, and found that the paired housing for one-month improved the learning and memory of the aging mice. These mice exhibited better performance on the Morris water maze (MWM) test, a longer freezing duration in the contextual fear conditioning test, a higher alternation rate in the T-maze test, and an increased preference for novel objects in the novel object recognition (NOR) test. The paired housing with young mice also improved the impaired long-term potentiation (LTP) in aging mice. CONCLUSIONS: Our results suggest that the paired housing with young mice has beneficial effects on learning and memory of aging mice. The manipulation of the systemic environment may, therefore, provide a new strategy for aging rejuvenation.

Long non-coding RNA SNHG1 contributes to cisplatin resistance in non-small cell lung cancer by regulating miR-140-5p/Wnt/ß-catenin pathway.

Small nucleolar RNA host gene 1 (SNHG1) has been identified to function as an oncogene in a large number of human cancers. Nevertheless, the biologic role and underlying molecular mechanism of SNHG1 on cisplatin (DDP)-resistance in NSCLC is still unknown. qRT-PCR assay was performed to assess the expression levels of SNHG1 and miR-140-5p. Western blot analysis was used to determine Wnt1, cyclinD1, c-Myc and ß-catenin levels. The direct correlation between SNHG1 and miR-140-5p was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. CCK-8 assay and Transwell assay were applied to determine cell proliferation ability, and cell migration and invasion capacities, respectively. Tumor xenograft was performed to confirm the effect of SNHG1 on DDP-resistance of NSCLC in vivo. Our data showed SNHG1 was upregulated in DDP-resistant NSCLC tissues and cell lines. SNHG1 knockdown suppressed the proliferation, migration, invasion and DDP-resistance in DDP-resistant NSCLC cell lines in vitro and inhibited tumor growth in vivo. Moreover, SNHG1 repressed miR-140-5p expression by directly binding to miR-140-5p. SNHG1-knockdown-mediated regulatory effect was antagonized by miR-140-5p. Furthermore, Wnt/ß-catenin signaling was involved in SNHG1/miR-140-5p-mediated regulation in DDP-resistance of NSCLC cell lines. The results suggested that SNHG1 knockdown ameliorated DDP-resistance of NSCLC by regulating miR-140-5p/Wnt/ß-catenin pathway, providing a new potential therapeutic target for DDP-resistance NSCLC treatment.

The potential application of plant wax markers from alfalfa for estimating the total feed intake of sheep.

Estimating the feed intake of grazing herbivores is critical for determining their nutrition, overall productivity and utilization of grassland resources. A 17-day indoor feeding experiment was conducted to evaluate the potential use of Medicago sativa as a natural supplement for estimating the total feed intake of sheep. A total of 16 sheep were randomly assigned to four diets (four sheep per diet) containing a known amount of M. sativa together with up to seven forages common to typical steppes. The diets were: diet 1, M. sativa + Leymus chinensis + Puccinellia distans; diet 2, species in diet 1 + Phragmites australis; diet 3, species in diet 2 + Chenopodium album + Elymus sibiricus; and diet 4, species in diet 3 + Artemisia scoparia + Artemisia tanacetifolia. After faecal marker concentrations were corrected by individual sheep recovery, treatment mean recovery or overall recovery, the proportions of M. sativa and other dietary forages were estimated from a combination of alkanes and long-chain alcohols using a least-square procedure. Total intake was the ratio of the known intake of M. sativa to its estimated dietary proportion. Each dietary component intake was obtained using total intake and the corresponding dietary proportions. The estimated values were compared with actual values to assess the estimation accuracy. The results showed that M. sativa exhibited a distinguishable marker pattern in comparison to the other dietary forage species. The accuracy of the dietary composition estimates was significantly (P < 0.001) affected by both diet diversity and the faecal recovery method. The proportion of M. sativa and total intake across all diets could be accurately estimated using the individual sheep or the treatment mean recovery methods. The largest differences between the estimated and observed total intake were 2.6 g and 19.2 g, respectively, representing only 0.4% and 2.6% of the total intake. However, they were significantly (P < 0.05) biased for most diets when using the overall recovery method. Due to the difficulty in obtaining individual sheep recovery under field conditions, treatment mean recovery is recommended. This study suggests that M. sativa, a natural roughage instead of a labelled concentrate, can be utilized as a dietary supplement to accurately estimate the total feed intake of sheep indoors and further indicates that it has potential to be used in steppe grassland of northern China, where the marker patterns of M. sativa differ markedly from commonly occurring plant species.

Effects of Nitrogen Application Rate on the Yields, Nutritive Value and Silage Fermentation Quality of Whole-crop Wheat.

Whole-crop wheat (Triticum aestivum L.) as forage has been extensively used in the world. In this study, the effects of N application rates on the yields, nutritive value and silage quality were investigated. The N application rates were 0, 75, 150, 225, and 300 kg/ha. The research results indicated that the dry matter yield of whole-crop wheat increased significantly with increasing N rate up to 150 kg/ha, and then leveled off. The crude protein content and in vitro dry matter digestibility of whole-crop wheat increased significantly with increasing N up to 225 kg/ha, while they no longer increased at N 300 kg/ha. On the contrary, the content of various fibers tended to decrease with the increase of N application. The content of lactic acid, acetic acid and propionic acid in silages increased with the increase of N rate (p<0.05). The ammonia-N content of silages with higher N application rates (≥225 kg/ha) was significantly higher than that with lower N application rates (≤150 kg/ha). Whole-crop wheat applied with high levels of N accumulated more nitrate-N. In conclusion, taking account of yields, nutritive value, silage quality and safety, the optimum N application to whole-crop wheat should be about 150 kg/ha at the present experiment conditions.

TM4SF5-CTD-2354A18.1-miR-4697-3P may play a key role in the pathogenesis of gastric cancer.

AIMS: Our aim is to identify important lncRNAs and mRNAs which may play a key role in contributing to pathogenesis of gastric cancer. METHODS: Different LncRNAs and mRNAs are identified by microarray in gastric cancer tissue and corresponding normal tissues. The function and relationship of different LncRNAs and mRNAs is performed by GO analysis and Pathway analysis and made code-non-code network (CNC) by Pearson correlation coefficients (PCC). Then mRNA-miRNA relationship is predicted through mRNA-miRNA relationship software (http://www.targetscan.org). Lastly, mRNA-miRNA-LncRNA network is established for further research. RESULTS: The expression profiles of 3732 lncRNAs showed different expression (fold change (FC)≥2.0, p<0.05) in gastric cancer tissue and normal tissue and expression profiles of 3994 mRNAs also showed different expression (FC≥2.0, p<0.05) in gastric cancer and corresponding normal tissue. CONCLUSION: The expression of TM4SF5, CTD-2354A18.1 and miR-4697-3P is in balance at physiological conditions, however, the balance is disrupted by some situations, which may contribute to gastric cancer. GO analysis and Pathway analysis also showed TM4SF5 played an important role in proliferation, differentiation and apoptosis. Therefore, TM4SF5-miR-4697-3P- CTD-2354A18.1 may play a key role in the pathogenesis of gastric cancer (Tab. 2, Fig. 4, Ref. 30).

Molecular characterization of tumor necrosis factor receptor-associated factor 6 (TRAF6) in pearl oyster Pinctada martensii.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key signaling adaptor molecule for tumor necrosis factor receptor superfamily and Toll-like receptor/interleukin-1 receptor family members. It signals the upstream receptors and is involved in a wide range of biological functions, such as immunity and bone metabolism. In this report, the TRAF6 gene from the pearl oyster Pinctada martensii (designated as PmTRAF6) was identified and characterized. The obtained full-length PmTRAF6 cDNA was 2273 bp, containing a 5'-untranslated region (UTR) of 297 bp, a 3'-UTR of 128 bp with a 42-bp poly (A) tail, and an open reading frame of 1848 bp that encoded 616-amino acid residues. The deduced protein sequence of PmTRAF6 contained a conserved TRAF family motif including a RING-type zinc finger, two TRAF-type zinc fingers, and a coiled-coil region followed by one meprin and TRAF homology domain. Multiple-sequence alignment indicated that TRAF6 was highly conserved among species, and PmTRAF6 showed 53% sequence identity to Azumapecten farreri and Mizuhopecten yessoensis. Furthermore, an amino acid sequence containing a low-complexity region was inserted in the TRAF6s from mollusk. Quantitative real-time polymerase chain reaction analysis demonstrated that PmTRAF6 was constitutively expressed in all tissues studied, with the most abundant mRNA expression in hepatopancreas and gill in P. martensii. After lipopolysaccharide stimulation, the expression of PmTRAF6 mRNA was dramatically upregulated. These results suggested that the obtained PmTRAF6 was a member of the TRAF6 family and perhaps involved in the innate immune response of pearl oyster.

Helicobacter pylori infection and normal colorectal mucosa-adenomatous polyp-adenocarcinoma sequence: a meta-analysis of 27 case-control studies.

AIM: The study aimed to determine whether Helicobacter pylori infection is associated with colorectal adenocarcinoma and to quantify the extent of the risk. METHOD: A literature search was performed to identify studies published between 1995 and 2012 for relevant risk estimates. Fixed and random effect meta-analytical techniques were conducted for colorectal adenocarcinoma and adenomatous polyp. RESULTS: Twenty-seven case-controlled studies involving 3450 adenocarcinoma patients, 1304 adenomatous polyp patients and more than 4000 controls were included. Helicobacter pylori was associated with an increased risk of colorectal adenocarcinoma and adenomatous polyp [odds ratio (OR) 1.24, 95% CI 1.12-1.37, P = 0.66; OR 1.87, 95% CI 1.53-2.28, P = 0.81]. There was a significant association between the CagA-positive strain and adenocarcinoma risk (OR 1.22, 95% CI 1.08-1.37, P = 0.05). In addition, there was an increased risk of tubular adenoma and villous adenoma formation (OR 3.06, 95% CI 1.98-4.73, P = 0.14; OR 2.05, 95% CI 0.84-4.97, P = 0.86). CONCLUSION: The meta-analysis suggests a promoting effect of Helicobacter pylori on the risk of adenocarcinoma. It also suggests that Helicobacter infection might have its influence at the start of the adenomatous polyp disease sequence.

Identification and characterization of a new type of asymmetrical dicentric chromosome derived from a single maternal chromosome 18.

Molecular cytogenetic analysis identified a new type of dicentric chromosome involving different breakpoints at 18q in a female fetus. The chromosome anomaly was designated as an asymmetrical pseudoisodicentric chromosome 18, 46,XX,psu dic(18)(pter-->q11.2::q21.3-->pter)mat. A series of BAC clones for 18q11.2 and q21.3 regions were used to identify one breakpoint within the region q11.2 between 19.8 and 21.6 Mb from the telomere of 18p and another breakpoint within q21.3 between 55.4 and 56.9 Mb from the telomere of 18p by FISH analysis. Real-time quantitative PCR and microsatellite analysis further verified that the dicentric chromosome was maternal in origin and resulted from a break-reunion between sister chromatids of a single maternal chromosome. We propose that a loop-type configuration of sister chromatids took place and that the break-reunion occurred at cross sites of the loop to form an asymmetrical isodicentric chromosome during either mitosis or meiosis. In this case, the asymmetrical pseudoisodicentric resulted in an 18pter--> q11.2 duplication and an 18q21.3-->qter deletion, which could have led to certain dysmorphic features of 18q- syndrome in this fetus.

Green luminescence band in ZnO: fine structures, electron-phonon coupling, and temperature effect.

The green emission band of ZnO has been investigated by both experimental and theoretical means. Two sets of equally separated fine structures with the same periodicity (close to the longitudinal optical (LO) phonon energy of ZnO) are well resolved in the low-temperature broad green emission spectra. As the temperature increases, the fine structures gradually fade out and the whole green emission band becomes smooth at room temperature. An attempt to quantitatively reproduce the variable-temperature green emission spectra using the underdamped multimode Brownian oscillator model taking into account the quantum dissipation effect of the phonon bath is done. Results show that the two electronic transitions strongly coupled to lattice vibrations of ZnO lead to the observed broad emission band with fine structures. Excellent agreement between theory and experiment for the entire temperature range enables us to determine the dimensionless Huang-Rhys factor characterizing the strength of electron-LO phonon coupling and the coupling coefficient of the LO and bath modes.

Resonant coupling of bound excitons with LO phonons in ZnO: excitonic polaron states and Fano interference.

We report on a photoluminescence observation of robust excitonic polarons due to resonant coupling of exciton and longitudinal optical (LO) phonon as well as Fano-type interference in high quality ZnO crystal. At low enough temperatures, resonant coupling of excitons and LO phonons leads to not only traditional Stokes lines (SLs) but also up to second-order anti-Stokes lines (ASLs) besides the zero-phonon line (ZPL). The SLs and ASLs are found to be not mirror symmetric with respect to the ZPL, strongly suggesting that they are from different coupling states of exciton and phonons. Besides these spectral features showing the quasiparticle properties of exciton-phonon coupling system, the first-order SL is found to exhibit characteristic Fano lineshape, caused by quantum interference between the LO components of excitonic polarons and the continuous phonon bath. These findings lead to a new insight into fundamental effects of exciton-phonon interactions.

Efficient multiphoton-absorption-induced luminescence in single-crystalline ZnO at room temperature.

At room temperature, multiphoton absorption- (MPA-) induced photoluminescence in ZnO strongly driven by a femtosecond (fs) near-infrared laser is studied. Two-photon absorption and three-photon absorption are proved to be responsible for the intense luminescence, when the wavelength of the fs excitation laser is above and below the half-bandgap of ZnO, respectively. Strong MPA absorption in ZnO is unambiguously evidenced by the interferometric autocorrelation measurements of the luminescence signal.

Predominant cellular immune response to the cartilage autoantigenic G1 aggrecan in ankylosing spondylitis and rheumatoid arthritis.

OBJECTIVES: Based on their HLA association, both ankylosing spondylitis (AS) and rheumatoid arthritis (RA) seem to be T-cell-driven diseases in which the autoantigens remain to be defined. One possible autoantigen is the G1 domain of aggrecan, the major cartilage proteoglycan. In BALB/c mice immunized with this protein, spondylitis and erosive polyarthritis have been reported. Immune reactivity to the G1 has been described in patients with RA and AS in an earlier study. Using novel and more sensitive techniques and relevant controls we sought to define the role of G1 as an autoantigen more precisely and to extend the specific analyses to the peptide level. METHODS: Peripheral blood (PB) mononuclear cells (MNC) from 47 AS patients, 22 RA patients and 20 healthy normal controls were exposed in vitro for 6 h to the cartilage-derived autoantigens G1, human cartilage (HC) gp-39 and collagen II. Synovial fluid (SF) MNC from seven AS and four RA patients were similarly analysed. Furthermore, PB MNC of 15 AS and 10 RA patients were examined with overlapping 18-mer peptides covering the whole G1 protein to identify the immunodominant epitopes. T cells were stained by monoclonal antibodies directed against the surface markers CD4, CD69 and against the intracellular cytokines interferon-gamma (IFN gamma), tumour necrosis factor-alpha (TNF alpha), interleukin 4 (IL-4) and IL-10. The percentage of reactive T cells was quantified by flow cytometry. RESULTS: After antigen-specific stimulation with the G1 protein, the CD4+ T cells of 30 AS patients (61.7%) and of 12 RA patients (54.5%) secreted significant amounts of IFN gamma and TNF alpha, while, in contrast, only 10% of the normal controls showed a response (P < 0.05). The synovial CD4+ T cells of five AS (71.5%) and of all four RA patients showed antigen-specific responses to the G1. In contrast, stimulation with HC gp-39 and collagen II showed no significant IFN gamma and TNF alpha secretion of MNC in all groups. Several G1-derived T-cell epitopes were identified as immunodominant in PB MNC of AS and RA patients and were partly overlapping. CONCLUSIONS: These data show that a cellular immune response to G1 is present in most AS and RA patients. G1-immunodominant epitopes were identified. The relevance of this finding for the pathogenesis of AS and RA remains to be established.

Molecular cloning of six novel Krüppel-like zinc finger genes from hematopoietic cells and identification of a novel transregulatory domain KRNB.

To clone zinc finger genes expressed in hematopoietic system, we designed primers based on conserved Cys(2)/His(2) zinc finger sequences to amplify corresponding domains from mRNA of normal bone marrow and leukemia cell line NB4. DNA fragments of novel zinc finger genes were chosen and used as probe pool to screen cDNA libraries or subject to rapid amplification of cDNA ends in order to obtain full-length cDNA. Six cDNAs including whole open reading frame of zinc finger proteins, named as ZNF191, ZNF253 (BMZF-1), ZNF255 (BMZF-2), ZNF256 (BMZF-3), ZNF257 (BMZF-4), and ZNF254 (BMZF-5) were obtained. All six belong to the Krüppel-like zinc finger gene family, and typical transcriptional regulatory motifs exist in the N-terminal moiety, such as the SCAN box in ZNF191, and the KRAB domains in ZNF253, ZNF254, ZNF256, and ZNF257. A previously undefined sequence nominated as Krüppel-related novel box, which may represent a new transregulatory motif, was revealed at the N terminus of ZNF255. The transregulatory function of non-zinc finger regions of ZNF191, ZNF253, and ZNF255 were addressed in yeast and mammalian cells. The results indicated that ZNF255 might be a conditional transactivator, whereas ZNF253 and ZNF191 displayed a suppressive effect on the transcription in yeast and/or mammalian systems.

[Assignment of a novel zinc finger gene ZNF191 to human chromosome 18Q12.1 by human/rodent somatic cell hybrid panel and fluorescent in situ hybridization].

A novel human zinc finger gene, ZNF191, was assigned to chromosome 18 by hybridization of human/rodent hybrid cell panel to a full-length cDNA as a probe. Meanwhile, a human genomic DNA lambda/DASH library was screened using this cDNA probe and several positive clones were obtained. Fluorescence in situ hybridization (FISH) was performed by using one of these positive clones, 16-1, as a probe. Thus, the ZNF191 gene was precisely mapped in 18q12. 1. To date, some hereditary diseases and tumors have been found to be associated with this region by analysis of genetic linkage and loss of heterozygosity. Hence, it suggested that the gene ZNF191 can be taken as a candidate gene responsible for those diseases and tumors.

Preliminary study on the loss of heterozygosity at 17p13 in gastric and colorectal cancers.

AIM: To evaluate the role of p53 in the development and progression of colorectal cancer and gastric carcinoma by analyzing the loss of heterozygosity (LOH) at 17p13.1 and 17p13.3. METHODS: LOH at the p53 gene locus and 17p13.3 were examined in 22 cases of gastric carcinoma and 14 cases of colorectal cancer by Southern blot analysis. RESULTS: Of the 22 gastrocarcinoma cases, 12 (54%) were heterozygous and LOH was detected in 6 (50%) of the 12 informative cases. In the 14 colorectal cancer cases, 10 (71%) were heterozygous, and LOH was detected in 6 (60%) of the 10 informative cases. CONCLUSION: LOH at the p53 gene locus is a frequent event in multiple step carcinogenesis progression. The high frequency of LOH at 17p13.3 suggests that there may be another tumor suppresser gene in that chromosome region.

Quantitative water mapping of cryosectioned cells by electron energy-loss spectroscopy.

A direct technique based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen-hydrated biological cryosections. Previously, methods for water determination have been indirect in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum-imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Reference spectra have been recorded from the major biomolecules (protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as +/- 3.5%. With the present instrumentation the maximum allowed dose of 2 x 10(3) e/nm2 limits the useful spatial resolution to around 80 nm for +/- 5% precision at a single pixel. By averaging pixel intensities a value of 56.8% with a precision of +/- 2.0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.

[45Ca-uptake, mitochondrial protein bound Ca2+ and ultrastructural distribution of Ca2+ in some brain regions of mice during drug-induced analgesia].

After buprenorphine (Bup, 0.8 mg/kg ip) treatment 45Ca-uptake (cpm/mg fresh brain) in vivo by brain slices decreased from 589 +/- 12 and 486 +/- 12 to 522 +/- 14 and 408 +/- 10 and mitochondrial protein bound Tb3+ (Tb3+ relative fluorescent intensity) reduced from 41 +/- 5 and 32 +/- 2 to 30 +/- 3 and 22 +/- 2 in periaqueductal grey and hypothalamus, respectively. A large amount dense precipitate occurred in the myelin sheath and mitochondria in both regions. The 45Ca-uptake evoked by buprenorphine at 16 micrograms/40 microliter in vitro has the similar tendency with that in vivo. Treated by ruthenium red (20 micrograms/mouse ip or icv) before buprenorphine, the above-mentioned effects were all abolished. Similar results were obtained with morphine (Mor, 10 mg/kg ip) and verapamil (Ver, 8 micrograms/mouse icv) instead of buprenorphine and ruthenium red, respectively. These results suggest that Ca2+ transport across neuroplasmic membranes plays a mediator role in drug-induced analgesia.