[Deletion of Aldh4a1 Leads to Impaired Sperm Maturation in Mice].

ALDH4A1, a member of the aldehyde dehydrogenase superfamily, is a key enzyme in the mitochondrial proline metabolism pathway. Recent studies have shown that mutations in aldh4a1 lead to reduced fertility and reproductive premature aging of male nematodes. However, the effect of ALDH4A1 on fertility of male mice has not been studied. In this study, we used CRISPR-Cas9 technology to construct a knockout mouse model of Aldh4a1 for the first time to explore the effect of this gene on the reproduction of male mice. The results showed that compared with WT male mice, Aldh4a1^(-/-) male mice were fertile, had normal spermatogenesis but defect in sperm maturation in the epididymis documented by impaired motility, increased morphological abnormalities and increased spontaneous acrosome reaction. In addition, transmission electron microscopy showed vacuoles in the sperm mitochondria, and fracture in the neck of sperms and vacuoles in these mice. These results revealed that ALDH4A1 plays a vital role in the structure of sperm flagellum and the process of sperm maturation in mice.

[Clinical manifestations and risk factors of poor prognosis of Acinetobacter baumannii intracranial infection].

Objective: To investigate the clinical features and risk factors for poor prognosis in patients with intracranial Acinetobacter baumannii infection. Methods: Retrospective analysis of data of patients who were hospitalized in the First Affiliated Hospital of AnHui Medical University between January 2011 and December 2017, and whose cerebral spinal fluid samples were positive for Acinetobacter baumannii and who were clinically demonstrated as intracranial infection during hospitalization was performed. Risk factors for poor prognosis were analyzed using single factor analysis and logistic regression analysis. Results: A total of 50 patients were included, with poor prognosis rate of 58% (29/50).92% of patients had history of craniotomy or operation of site adjacent to brain.Major type of intracranial infection was purulent meningitis.Fever rate was 100%.Multidrug resistant Acinetobacter baumannii accounted for 89.01%. Sensitivity to meropenem was only 9.09%.Shock, multi-drug resistant bacteria and no intrathecal injection were risk factors for poor prognosis of patient.Multi-factor logistic regression analysis showed "no intrathecal injection" was independent risk factor for poor prognosis of patients with intracranial Acinetobacter baumannii infection. Conclusions: Acinetobacter baumannii that induced intracranial infection is mostly highly drug-resistant bacterium, with high risk of post-infection poor prognosis.Clinically, it is essential to take proper peri-operative measures and early identify occurrence of intracranial infection.Reasonable application of anti-infection drug and external ventricle drainage, especially intrathecal injection of aminoglycosides, can be promoted as a kind of safe and effective means.

[Effect of cigarette smoke exposure during pregnancy on offspring of ovarian development and oocyte DNA methylation in female mice].

Objective: To explore the effects of cigarette smoke exposure during pregnancy on the quality of oocytes and the whole genome DNA methylation in the offspring of female mice during the period of germinal vesicle (GV) . Methods: The pregnant 7 d mice were divided into 3 groups, exposure on the 0, 0.5, 1 lit cigarettes cabinet (volume 18 L) , at 9 am and 3 pm for 1 h twice daily, until delivery. When the mice were 6 weeks old, the organ index and the number of follicles in the ovary were detected by weighing and making HE stained sections. GV stage oocytes were obtained by Hoechst 33342 staining and indirect immunofluorescence to detect the quality of oocytes, chromatin configuration and whole genome DNA methylation level. Results: Compared with the control group and low dose group, the offspring ovarian organ index of female mice in the high dose group decreased, the difference was statistically significant (P<0.05) . Compared with the control group, the number of follicular oocytes in low dose group and high dose group female mice offspring decreased, the zona pellucida oocytes diameter decreased, and the zona pellucida thickness increased, the differences were statistical significance (P<0.05) . Compared with control group and low dose group, in the high dose group, the oocytes nucleus diameter of the female mice offspring decreased, the proportion of nucleolus surrounded type chromatin configuration (SN) decreased, and the proportion of nucleoil not surrounded type (NSN) increased, the relative fluorescence intensity of 5MeC decreased, the differences were statistically significant (P<0.05) . Conclusion: Exposure to cigarette smoke during pregnancy may reduce indicators of offspring ovarian organ index, female rat follicle number and oocyte quality change, the high dose group can lead to oocyte chromatin structure and DNA methylation level anomaly.

Isolation and characterization of atrazine-degrading strain Shewanella sp. YJY4 from cornfield soil.

UNLABELLED: Atrazine has been used worldwide for over 50 years as a chemical herbicide. A strain of bacteria, YJY4 which utilizes atrazine as its sole nitrogen source for growth was isolated from agricultural black soil in northeastern China. 16S rDNA sequencing identified YJY4 as a Shewanella sp. PCR analysis and sequencing confirmed that YJY4 contained atrazine-degrading atzA, atzB and atzC genes. These genes revealed high similarity with those in Pseudomonas sp. ADP and Arthrobacter sp. TC1. The strain YJY4 was observed to degrade atrazine (100 mg l(-1) ) to cyanuric acid completely after 36 h. To the best of our knowledge, YJY4 was the first reported Shewanella sp. to grow in pure culture with atrazine serving as a sole source of nitrogen. Therefore, YJY4 may help with atrazine biodegradation and may become an abundant resource of atrazine degradation strains. SIGNIFICANCE AND IMPACT OF STUDY: A new isolated strain, YJY4 showed high atrazine-degrading ability, being able to degrade 100 mg l(-1) atrazine completely in 36 h. Strain YJY4 was identified as Shewanella sp. and contained atrazine-degrading atzA, atzB and atzC genes. This study examined the degradation mechanism and metabolic ability of this strain and for the bioremediation of contaminated environments, provides more strain selection and determines the strain of atrazine bioremediation potential.

Differences in H3K4 trimethylation in in vivo and in vitro fertilization mouse preimplantation embryos.

Trimethylation of lysine 4 at histone 3 (H3K4me3) is considered a marker of active transcription; it plays an important role in transcription reprogramming efficiency. We compared the levels of H3K4me3 in mouse preimplantation embryos from MII stage oocytes produced by in vivo and in vitro fertilization (IVF) using immunofluorescence histochemistry. IVF embryos were further treated with trichostatin A (a histione deacetylase inhibitor) to investigate the effect of histone acetylation on H3K4me3. We found higher levels of H3K4me3 in MII stage oocytes in metaphase chromosomes. The pattern of H3K4 trimethylation of in vivo embryos from zygote to blastocyst stages was similar to that of IVF embryos; however, the concentration of H3K4me3 was significantly higher in the in vivo fertilization embryos. The levels of H3K4me3 in the trichostatin A-treated groups were also significantly increased. We conclude that culture condition and environmental changes can cause histone modification and that the effect of these environmental conditions on epigenetic changes should be taken into consideration.

Characteristics of DNA repair induced by DNA polymerase ß in hepatoma cells after γ-ray irradiation.

AIM: To investigate the effects of DNA repair induced by DNA polymerase ß in hepatoma cells after γ-ray irradiation. METHODS: Cell nuclei were prepared from mouse model (SMMC LTNM), in which human hepatoma cells are transplanted on nude mice. The nuclei were then irradiated with (60)Co-γ rays at different dose levels or dose rates. A selective inhibitor test was then used to detect the effects of the radiation on DNA repair using N-ethylmaleimide (NEM) and ddTTP as selective inhibitors to DNA polymerases γ and ß respectively. RESULTS: (3)H-TTP incorporation into irradiated nuclei or calf thymus DNA was significantly higher than that the rate at which it is incorporated into non-irradiated nuclei when either DNA polymerase ß or γ was inhibited. When both NEM and ddTTP are present, the (3)H-TTP incorporation in irradiated DNA was not significantly different from the non-irradiated nuclei. Furthermore, (3)H-TTP incorporation into DNA of SMMC-LTNM hepatoma nuclei was higher than that of normal hepatocyte nuclei (P < 0.01). This suggests that DNA repair induced by DNA polymerase ß was more active in hepatoma cell nuclei than in normal hepatocyte nuclei. CONCLUSION: DNA polymerase ß may be more responsive to DNA damage in some tumor cells than that in normal cells, which may facilitate the cells to repair DNA damages from radiation more efficiently.

Progestin and antiprogestin differentially regulate the expression of insulin-like growth factors (IGF-I and IGF-II) messenger ribonucleic acid in human endometrial stromal cells.

Previous studies have shown that insulin-like growth factors (IGF-I and IGF-II) stimulate mitogenic activity in human endometrial stromal cells. In the present study, we have investigated the expression of IGF-I and -II mRNA to ascertain any autocrine growth-promoting effect in this system. Northern blot analysis revealed that endometrial stromal cells express multiple sizes of IGF-I and -II transcripts. The effect of progestin and antiprogestin was studied during decidualization of endometrial stromal cells in long-term culture. Solution hybridization and a ribonuclease protection assay of control cells revealed that the level of IGF-I mRNA was low, whereas IGF-II mRNA was always abundant. Medroxyprogesterone acetate (MPA) stimulated the expression of IGF-I mRNA > 4-fold in predecidualized cells during the first 10 days of culture. IGF-I mRNA decreased to basal level in prolonged culture when cells were decidualized. In contrast, MPA suppressed the IGF-II mRNA level by 60% in predecidualized cells, but IGF-II mRNA was highly expressed after 20 days of incubation with MPA (5-fold increase from Days 5-10 to Day 20 of culture). In progestin-pretreated cells, addition of the antiprogestin RU486 for 1-4 days reduced IGF-I mRNA by 50-90%. RU486 reversed the suppressive effect of MPA and increased IGF-II mRNA. This study indicates that progestin and antiprogestin differentially regulate IGF-I and IGF-II mRNA levels in human endometrial stromal cells.

[The intermediate filament-lamina-nuclear matrix system of ES-M13 cells].

Sequential extraction and DGD embedment-free section TEM techniques were used as new methods to study the intermediate filament-lamina-nuclear matrix (IF-L-NM) system of cells. Murine embryonic stem cells (ES-M13) were investigated by means of electron microscopy, immunofluorescence microscopy and Western-blot analysis. There existed a delicate nuclear matrix network in the nucleus domain of detergent-extracted ES cells. The filaments of the nuclear matrix were found in close association with the nuclear lamina. Some intermediate filaments were also observed in the cytoplasm. In immunofluorescence microscopy, a bright, slightly granular cytoplasmic fluorescence, showing no polar concentration, was revealed with keratin monoclonal antibody AF5. We could not detect any significant fibrillar staining in the ES cells by indirect immunofluorescence method. With antibodies to vimentin and desmin, the ES cells showed only a nonspecific, weak fluorescence, similar to that seen in the control. In Western-blot analysis of electrophoretically separated polypeptides, three polypeptides with molecular weight of 65 KD, 62 KD and 52 KD, reactive with keratin monoclonal antibody were detected in the ES cells.

Identification of a distal regulatory sequence of the human IGFBP-1 gene promoter and regulation by the progesterone receptor in a human endometrial adenocarcinoma cell line.

The activity of the insulin-like growth factor binding protein-1 (IGFBP-1) gene promoter was studied in the human endometrial adenocarcinoma cell line HEC-1B. Basal promoter activity was directed by the region +68 to -207 bp, similar to observations in the hepatoma HepG2 cell line. A distal regulatory sequence approximately -2.6 kb from the transcription initiation site strongly enhanced the activity of the IGFBP-1 gene promoter in HEC-1B cells, but not in HepG2 cells. Sequence analysis revealed that this active region resides in 105 bp between -2,628 to -2,732 bp (the Rsa I-Cla I fragment). This region contains many putative active motifs homologous to known cis elements. Additional deletion and mutation in the Rsa I-Cla I fragment showed that the activity was confined to a 58-bp DNA fragment. In cells treated with progestin and co-transfected with progesterone receptor vector hPR1, the CAT activity derived from constructs containing the Rsa I-Cla I fragment was reduced in a dose-dependent manner. The active DNA fragment also stimulated the activity of the heterologous TK/CAT promoter in HEC-1B cells, while the PR complex inhibited this activity by 50%. These observations indicate that most of the regulation of the IGFBP-1 gene in HEC-1B cells is derived from the distal promoter region confined to the Rsa I-Cla I fragment and that the same region mediates an inhibitory effect from the progesterone receptor.

Activation of the human IGFBP-1 gene promoter by progestin and relaxin in primary culture of human endometrial stromal cells.

We have studied the activity for the insulin-like growth factor binding protein-1 (IGFBP-1) gene promoter in human endometrial stromal cells by transient transfection. The promoter activity derived from p3.6CAT or p3.6Luc (3400 bp IGFBP-1 promoter 5' to the reporter gene chloramphenicol acetyltransferase or luciferase) was minimal in unstimulated cells. A time study over 13 days of culture showed that the promoter activity increased exponentially to > 10(4) fold in cells treated with medroxyprogesterone acetate (MPA) and relaxin (RLX). Induction of the IGFBP-1 gene promoter activity by hormones was similar to the secretion pattern of IGFBP-1 in endometrial stromal cells. MPA alone caused a moderate induction, 3-40-fold increase over the control. Deletion analysis showed that two regions in the IGFBP-1 gene promoter were responsible for the activation of the IGFBP-1 gene. The basal promoter region, termed bp1-A (+68 bp to -1.205 kb), contains multiple sections of regulatory sequence including a cis-element CCAAT (-72 bp). A DNase I protection assay in the bp-1A region revealed four distinct binding regions, one of which contained the CCAAT box region. Another promoter region, termed bp1-B (-2.6 to -3.4 kb), mediated 95% of the total promoter activity in endometrial stromal cells. The bp1-B region also contains multiple regulatory sequences. Mutation and DNase I protection assay suggest that Sp1-like binding site at -2.63 kb was a regulatory site responsible for the activation of IGFBP-1 gene promoter.

Effect of progestin, antiprogestin, and relaxin on the accumulation of prolactin and insulin-like growth factor-binding protein-1 messenger ribonucleic acid in human endometrial stromal cells.

Prolactin (PRL) and insulin-like growth factor-binding protein (IGFBP-1) are two major secretory proteins of human endometrial/decidual cells. We have characterized the mRNA of PRL and IGFBP-1 and studied the effect of progestin, medroxyprogesterone acetate (MPA), anti-progestin (RU486), and relaxin (RLX) on the levels of these two mRNA transcripts in a long-term culture of human endometrial stromal cells. Northern blot analysis showed that the size of PRL mRNA was 1.15 kb and that of IGFBP-1 mRNA, 1.6 kb. Primer extension of endometrial/decidual IGFBP-1 mRNA showed two transcription initiation sites identical to those found in HepG2 human hepatoma cell line. The levels of mRNA in control samples remained low, approximately 2 pg PRL and approximately 5 pg IGFBP-1/microgram RNA at various times of culture. When stromal cells were treated with MPA for 28 days, PRL mRNA gradually increased 100-fold whereas IGFBP-1 mRNA exponentially increased approximately 1000-fold compared to control values and leveled after 25 days in culture. The timing of maximal stimulation was shortened by withdrawing MPA or by replacing MPA with RU486. After removal of MPA, levels of both mRNAs increased and each peaked after approximately 10 days, with PRL showing a 2-fold and IGFBP-1 a 20-fold increase compared to cells treated with MPA continuously. Replacing MPA by RU486 caused a rapid increase of PRL mRNA (2-3-fold) in 2-3 days followed by a gradual reduction to less than 20% of peak levels over the next 3 days. IGFBP-1 mRNA levels increased 30- and 100-fold in 1-2 days followed by a reduction to less than 20% of peak levels over the next 24 h. The reduction of mRNA levels by RU486 was reversed when cells were rechallenged with MPA. Relaxin alone caused a transient stimulation of PRL and IGFBP-1 mRNA. Maximal stimulation occurred between 10 and 20 days of culture and was 100-fold for PRL and 1000-fold for IGFBP-1 relative to control values. Cells treated with MPA and RLX in sequence had higher mRNA levels than cells treated with MPA continuously or cells subjected to MPA withdrawal. Maximal mRNA levels reached 0.4 ng PRL and approximately 8 ng IGFBP-1/microgram total RNA, approximately 0.04% and 0.8% of cellular RNA. The mRNA levels under various hormonal manipulations were similar to the previously published synthesis and secretion patterns of PRL and IGFBP-1 proteins in this system.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal autotransplantation with A-V anastomosis for treatment of Cushing's disease. A follow-up study of 31 cases.

Thirty-one patients with Cushing's disease were treated with bilateral adrenalectomy and partial left adrenal autotransplantation with A-V anastomosis. They were followed up for 1 to 5 years. Three patients failed after adrenal autotransplantation to the abdominal muscle with the left gland's central vein anastomosed to the inferior epigastric artery. Twenty-eight patients underwent adrenal autotransplantation to the omentum with the gland's vein anastomosed to the gastroepiploic artery. Steroid could be omitted a short period after operation and life-long replacement therapy was avoidable in most patients. A few patients subsequently developed hypocorticalism. Two died and one had recurrence of Cushing's disease. Clinical and animal experimental results showed that the omentum is suitable for adrenal autotransplantation with A-V anastomosis. Transabdominal bilateral adrenalectomy and adrenal autotransplantation to the omentum with A-V anastomosis are acceptable in the treatment of Cushing's disease. The subsequent development of hypocorticalism and recurrence of Cushing's syndrome are also discussed.

Activation of the trpBA promoter of Pseudomonas aeruginosa by TrpI protein in vitro.

We have developed an in vitro transcription system in which purified TrpI protein and indoleglycerol phosphate (InGP) activate transcription initiation at the trpBA promoter (trpPB) and repress initiation at the trpI promoter (trpPI) of Pseudomonas aeruginosa. The phenotypes resulting from mutations in the -10 region of both promoters indicate that the -10 region consensus sequence in P. aeruginosa is probably the same as that in Escherichia coli. Furthermore, in the absence of TrpI and InGP, the activities of the two promoters are inversely correlated: down mutations in trpPI lead to increased activity of trpPB, and up mutations in trpPB cause a decrease in trpPI activity. These results are a consequence of the fact that the two promoters overlap, so that RNA polymerase cannot form open complexes with both promoters simultaneously. Thus, in theory, by preventing RNA polymerase from binding at trpPI, TrpI protein could indirectly activate trpPB. However, oligonucleotide-induced mutations that completely inactivate trpPI do not relieve the requirement for TrpI and InGP to activate trpPB. Therefore, activation of trpPB is mediated by a direct effect of TrpI on transcription initiation at trpPB. In addition, the oligonucleotide-induced mutations in trpPI alter site II, the weaker of two TrpI binding sites identified in DNase I and hydroxyl radical footprinting studies (M. Chang and I. P. Crawford, Nucleic Acids Res. 18:979-988, 1990). Since these mutations prevent full activation of trpPB, we conclude that specific base pairs in site II are required for activation.

RNA polymerases from Pseudomonas aeruginosa and Pseudomonas syringae respond to Escherichia coli activator proteins.

The activities of RNA polymerases (RNAPs) from Pseudomonas aeruginosa and Pseudomonas syringae were compared with that of Escherichia coli RNAP. All three enzymes are able to initiate transcription at the trpBA promoter of P. aeruginosa and at the coliphage lambda promoters, pRM and pRE, in response to heterospecific activators (TrpI protein, repressor, and cII protein, respectively). However, both Pseudomonas polymerases have less stringent requirements for promoter recognition in the absence of activators than does E. coli RNAP.

[Distribution and metabolism of calcium [3H]metronidazolamate in mice].

The blood radioactivity-time curve after iv calcium [3H]metronidazola-mate in mice was to be a diexponential model. It was rapidly distributed in various tissues. The highest radioactivities were found in liver and kidney, followed by lung, blood, heart, spleen, and the lowest in brain and testes. Bile and bone marrow contained only a slight radioactivity. In 7 d the cumulative excretion of radioactivity was 52 +/- 17% of the iv dose in urine and 10.3 +/- 2.4% in feces. The measurement by TLC and liquid scintillation counting of urine taken at 24 h after iv calcium [3H] metronidazolamate to mice revealed that approximately 77% of the total radioactivity in urine was excreted as the drug in unchanged form and 7% as its hydrolysate, metronidazole. Calcium [3H]metronidazolamate remained at a high level in blood on account of its longer T1/2 beta (34 h) and 20% were bound to plasma protein, thus making it available for longterm fractional radiotherapy of tumors.

[Primary structure of the leading sequence of 16S rRNA gene in Brassica napus chloroplast].

The 840bp leading sequence of 16S rRNA gene (including 140bp 16S rRNA gene) was sequenced. Screening for structure elements common to tRNA reveals a gene coding for tRNA(Val). An open reading frame, and three E. coli RNA polymerase binding sites were found. In front of the open reading frame, the tRNA(Val) gene, and the 16S rRNA gene, a stable stem-loop structure can be formed. We suggested that these stem-loop structures may have some effect on the gene transcription which are located on the inverted repeat sequence in chloroplast.

[The complete nucleotide sequence of Brassica napus chloroplast 16S rRNA gene].

The complete nucleotide sequence of a 16S rRNA gene and its flanking sequence from Brassica napus chloroplast has been determined. The 16S rRNA gene is 1491 bp in length, having 98.5%, and 96.1% homology with those of tobacco and maize chloroplast, respectively. The large stem and loop structure can be constructed from the sequence surrounding the 5' and 3' ends of the 16S rRNA gene. The stem is about half shorter than that of tobacco, due to a 79 bp deletion in Brassica napus chloroplast.