LncRNA NBR2 inhibits EMT progression by regulating Notch1 pathway in NSCLC.

OBJECTIVE: The aim of this study was to identify the role of long non-coding RNA (lncRNA) NBR2 in non-small-cell lung cancer (NSCLC) and its possible molecular mechanisms. PATIENTS AND METHODS: The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to explore lncRNA NBR2 expression in NSCLC cells and tissues. The chi-square test was used to analyze the relationship between lncRNA NBR2 expression and the clinical features of NSCLC patients. The pcDNA3.1 and pcDNA3.1-NBR2 vectors were transfected into NSCLC cells, and the proliferation and migration ability of NSCLC cells were detected using cell counting kit-8 (CCK-8) and transwell assay. The epithelial-mesenchymal transition (EMT)-related genes expression was detected by an EMT RT2 PCR array. QRT-PCR and Western blot was used to analyze the mRNA and protein levels of Notch1, Vimentin, N-cadherin, E-cadherin, HEY1, HEY2, and HEYL. RESULTS: The expression of lncRNA NBR2 was decreased in NSCLC patients tissues, and the NSCLC patients in the NBR2 low expression group showed a poor prognosis. Meanwhile, the expression of NBR2 in patients with NSCLC was correlated with tumor size. Overexpression of NBR2 suppressed the viability and migration of NSCLC cells and the expression of Notch1 and EMT-related genes in AsPC-1 cells. Simultaneous overexpression of NBR2 and Notch1 could reverse the inhibitory effect of NBR2 on proliferation and migration of NSCLC cells. CONCLUSIONS: LncRNA NBR2 inhibited the progression of EMT in NSCLC by regulating the Notch1 pathway.

[Association of polycyclic aromatic hydrocarbon exposure and CYP1A1 gene and enzyme activity of peripheral blood monocyte cells in coke oven workers].

Objective: To explore the association between polycyclic aromatic hydrocarbons (PAHs) exposure and cytochrome P450 CYP1A1 expression at gene and enzyme activity levels in the peripheral blood monocyte cells in coke oven workers, and to provide a certain basis for the biological monitoring of health damage in coke oven workers. Methods: We surveyed 118 coke oven workers and 63 controls (energy power workers in the same company) using self-designed questionnaire, determined their post-shift urinary 1-hydroxypyrene (1-OH-Py) concentration using high performance liquid chromatography (HPLC)-fluorescence detector method. We also isolated the peripheral blood mononuclear cell (PBMC) from fasting venous blood, and detected DNA damage using comet assay, CYP1A1 mRNA level using the real-time fluorescent quantitative PCR (FQ-PCR), and EROD activity using spectrophotometry. Statistical analyses including one-way analysis of variance and multiple linear regressions were used to analyze the association of urinary 1-OH-Py and CYP1A1 mRNA level and EROD activity. Results: Compared to the control group, the urinary 1-OH-Py concentration and PBMC DNA tail moment were significantly increased in coke oven workers (P<0.05), and CYP1A1 gene level and EROD activity in PBMC were significantly decreased (P<0.05). Multiple linear regression showed that a ten-fold increase of urinary 1-OH-Pycon centration was associated with a decrease of 0.77 (95%CI: -1.33--0.21) in CYP1A1 gene level, and a decline of 0.15 (95%CI: -0.76--0.16) in EROD activity of PBMC in coke oven workers (P<0.05). Conclusion: Occupational PAHs exposure induced DNA damage, which was associated with the decreased level in CYP1A1 gene cavel and EROD activity in PBMC of coke oven workers.

[Evaluation on the effect of exclusive breastfeeding among women with primipara, using the Information-motivation-behavioral skills model intervention model].

Objective: To explore the effects of intervention programs during primipara, using the Information-motivation-behavioral skills model (IMB) intervention model on initial exclusive breastfeeding. Method: 206 women of primiparas who were registered to the top three hospitals were selected in Shandong province, 2018. These women were hospitalized in the obstetrics/gynaecology wards during the later periods. IMB model was used to support the intervention programs at the following stages: antenatal period, 1 day postpartum, 3-4 days postpartum and 42 days postpartum. Questionnaire regarding self-efficacy energy meter, knowledge and behavior related to breastfeeding, were used before and after the intervention programs. Results: Through intervention programs, statistically significant differences in breastfeeding self-efficacy scores between the four stages (F=150.680, P=0.000) were recorded. Significant differences were also noticed in scores on knowledge of breastfeeding (F=176.556, P=0.000). Conclusion: The application of IMB intervention model in women at childbearing age seemed to be helpful in improving the effect of primary breastfeeding in women at childbearing age.

Effects of organic zinc on tibia quality, mineral deposit, and metallothionein expression level of aged hens.

The study aimed to determine the effects of methionine hydroxy analog chelate zinc on the tibia quality, mineral deposit, apparent retention of nutrients, and liver metallothionein (MT) expression level of aged laying hens. A total of 960 layers (Hy-Line Grey, 57 wk old) were randomly assigned into 4 groups, and each group had 8 replicates of 30 hens. During the first 2 wk, groups were fed a basal diet without extra zinc (Zn: 35.08 mg/kg). During the ensuing 14 wk, 4 levels of Zn (inorganic Zn: 80 mg/kg; organic Zn: 20, 40, 80 mg/kg) were added to the diet. The results indicated that both the Zn source and level did influence tibia strength and calcium (Ca) and Zn concentrations of tibia (P < 0.05), whereas there were no differences in the copper (Cu) and phosphorus (P) concentrations of the tibia and the tibia length (P > 0.05). Moreover, dietary supplementation with 40 or 80 mg/kg of organic Zn showed higher Zn and Ca concentrations in the tibia and higher tibia strength. The Cu concentration in the liver showed no difference among the 4 treatments, whereas the Zn concentration in the liver increased with the increasing Zn level. The apparent retention of P, iron (Fe), and manganese (Mn) was not affected by the Zn level or source (P > 0.05). However, the organic Zn group increased the apparent retention of Cu, Zn, Ca, crude protein (CP), and energy, and the group supplemented with 40 or 80 mg/kg of organic Zn obtained significant effects (P < 0.05). Moreover, dietary supplementation with 40 or 80 mg/kg organic Zn increased the MT mRNA expression of the liver at week 72, whereas 20 mg/kg of organic Zn decreased it (P < 0.05). In conclusion, this study suggested that an optimum dietary (40 mg/kg) organic Zn level plays a key role in promoting the apparent retention of minerals and nutrients, trace element deposit, and MT mRNA expression.

MiR-411-5p acts as a tumor suppressor in non-small cell lung cancer through targeting PUM1.

OBJECTIVE: The aim of this study was to investigate the expression of micro ribonucleic acid-411-5P (miR-411-5p) in non-small cell lung cancer (NSCLC), and to explore the effect of miR-411-5p on the biological behavior of NSCLC cells as well as the underlying molecular mechanism. PATIENTS AND METHODS: Quantitative Real Time- Polymerase Chain Reaction (qRT-PCR) was used to detect the expression level of miR-411-5p in NSCLC tissues and cells. MiR-411-5p mimics and relevant controls were transfected into NSCLC cells according to the instructions of Lipidosome 2000. Transfected cells were divided into the experimental group and the control group. The transfection efficiency of each group was detected by qRT-PCR. After miR-411-5p overexpression, methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, and transwell assay were used to detect the biological changes of cells in each group. Bioinformatics predicted that pumilio homolog 1 (PUM1) was the target gene of miR-411-5p. Subsequently, the mRNA and protein expression level of PUM1 in each group was detected by qRT-PCR and Western blotting, respectively. Dual-luciferase reporter assay was used to validate the target regulatory relationship between miR-411-5p and PUM1. RESULTS: The results of qRT-PCR showed that miR-411-5p was relatively lowly expressed in NSCLC tissues and cells. After miR-411-5p overexpression, MTT results revealed that the proliferation of NSCLC cells was decreased. Flow cytometry results indicated that the apoptosis rate of NSCLC cells was increased, and cell cycle was arrested in the G0-G1 phase. Meanwhile, the transwell assay demonstrated that the migration and invasion abilities of NSCLC cells were decreased. Bioinformatics predicted that PUM1 was the target gene of miR-411-5p. After miR-411-4p was overexpressed in NSCLC cells, qRT-PCR and Western blotting showed that both the mRNA and protein expression levels of PUM1 were up-regulated. Moreover, dual-luciferase reporter assay demonstrated that miR-411-5p could significantly inhibit the luciferase activity of wild-type PUM1-3'-untranslated region (3'-UTR). However, it exhibited no effect on the luciferase activity of cells transfected with mutant plasmids. CONCLUSIONS: MiR-411-5p may be involved in regulating the biological function of NSCLC cells via targeting PUM1. In addition, miR-411-5p may serve as a potential target for the molecular therapy of NSCLC.

Effects of methionine hydroxyl analog chelated zinc on laying performance, eggshell quality, eggshell mineral deposition, and activities of Zn-containing enzymes in aged laying hens.

The study aimed to determine the effects of methionine hydroxyl analog chelated zinc (MHA-Zn) on laying performance, eggshell quality and mineral deposits, and the activities of Zn-containing enzymes on aged laying hens. A total of 960 layers (Hy-Line Grey, 57 wk old) were fed a basal diet (Zn: 35.08 mg/kg) without extra zinc for 2 wk. During the ensuing 14 wk, birds were randomly divided into 4 groups according to body weight and laying rate, with 8 replicates per treatment, and each group had 8 replicates of 30 hens. Four levels of Zn (ZnSO4: 80 mg/kg; MHA-Zn: 20, 40, 80 mg/kg) were added to the diet, respectively. The results shown that dietary Zn did not affect laying rate, average egg weight, average daily feed intake, or feed conversion ratio (P > 0.05); however, compared to the inorganic group, dietary supplementation with 40 or 80 mg/kg MHA-Zn decreased broken egg rate significantly in the whole period (P < 0.05), while significantly increased eggshell weight in week 62 to 72, eggshell thickness and eggshell strength in wk 66 to 72, eggshell weight percent and eggshell density in week 62 to 72 (P < 0.05). Besides, dietary supplementation with different sources and levels of Zn did not affect ash concentration of eggshell (P > 0.05), whereas dietary supplementation with 80 mg/kg MHA-Zn improved the Zn and Ca concentrations of eggshells and carbonic anhydrase (CA) activity of liver, and 40 mg/kg MHA-Zn increased Zn concentration of liver (P < 0.05). Moreover, no significant differences in alkaline phosphatase activity were observed among the treatment groups (P > 0.05). Therefore, dietary supplementation with 40 mg/kg MHA-Zn can improve eggshell quality by promoting Ca deposition and CA activity.

Dietary methionine requirement of Jing Brown layer hens from 9 to 17 weeks of age.

This study was conducted to evaluate the effect of dietary methionine (Met) supplementation in growth performance and reproductive performance of Jing Brown layer hens. A total of 375 9-week-old Jing Brown layer hens were allocated equally to five treatments consisting of 5 replicates with 15 hens. Hens were fed with a diet of corn and soya bean meal supplemented with 0.23%, 0.27%, 0.31%, 0.35% and 0.39% Met respectively. Different Met levels did not significantly affect average daily feed intake (ADFI), average daily gain (ADG) and feed/gain ratio (F/G) (p > 0.05), whereas flock uniformity (FU) and jejunum index were significantly different (p < 0.05), and the largest FU was observed in 0.31% Met. Dietary supplementation of Met significantly affected reproductive system development (p < 0.05), and 0.27-0.31% Met obtained optimal reproductive system development. Different Met levels significantly affected serum uric acid and alkaline phosphatase. Moreover, the relatively higher reproductive hormones in serum were observed in 0.27% Met. Analysis of quadratic curve estimation of flock uniformity, the total number of follicles, the primary follicles and the secondary follicles showed that the optimal Met levels were 0.293%, 0.286%, 0.286% and 0.288%, which could be averaged to 0.288%. These results suggested that the optimal Met requirement for Jing Brown layer hens from 9 to 17 weeks old is 0.29%.

Effects of dietary threonine levels on growth performance, serum biochemical indexes, antioxidant capacities, and gut morphology in broiler chickens.

A dose-response experiment was conducted to investigate the impacts of dietary threonine (Thr) levels on growth performance, serum biochemical indices, antioxidant capacities, and gut morphology of broiler chickens. Four hundred and thirty-two 1-d-old commercial broilers were allocated to 4 treatments consisting of 6 replicates of 18 birds. The experimental treatments received the same Thr-deficient basal diet and were labeled as follows: 85%, 100%, 125%, and 150% of NRC (1994) recommendations. The results demonstrated that on 21 d and 42 d, average daily weight gain (ADG, 22 to 42 d, 0 to 42 d) increased quadratically or cubically as the inclusion of Thr increased, while feed conversion ratio (FCR, 0 to 21 d, 0 to 42 d) decreased quadratically or cubically as dietary Thr increase from 85% to 150%. Excess dietary Thr levels triggered plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities. The concentrations of total protein (TP) and globulin (GLO) increased quadratically with increasing Thr level, and the highest concentrations of TP and GLO were obtained at the 125% Thr level. Moreover, the plasma uric acid (UA) concentration decreased linearly or quadratically with the increase in dietary Thr level. Likewise, the serum glutathione peroxidase (GSH-Px) and total superoxide dismutases (T-SOD) activities increased quadratically as dietary Thr increased, and the highest activity of GSH-Px was obtained at the 125% Thr level, while the highest T-SOD level occurred in the 100% Thr group. Gut morphology of birds showed significant response to different graded concentrations of Thr level. Villus height (VH), crypt depth (CD), and VH:CD ratio (VH/CD) were increased linearly or quadratically by Thr supplementation. Therefore, the present study suggests that the NRC (1994) recommendations Thr level that was optimum for growth performance, and 125% of the NRC (1994) recommendations Thr level had better effects on biochemical indices, antioxidant function, and gut morphology of broilers.

Effects of dietary supplementation of synbiotics on growth performance, intestinal morphology, sIgA content and antioxidant capacities of broilers.

Today, several strategies are being used to decrease the serious effects of antibiotics abuse on broilers industry and public health, among which synbiotics are one of the most promising antibiotic alternative. This study was undertaken to assess the effects of synbiotics, which composed of probiotics (Bacillus subtilis) and prebiotics (xylooligosaccharide and mannanoligosaccharide), on growth performance, intestinal morphology, sIgA content and antioxidant parameters of broilers. Four hundred and fifty one-day-old commercial Cobb48 broilers were assigned to five treatments consisting of six replicates of 15 birds each pen. Five dietary treatments include basal diets (control), basal diets plus antibiotics (4 mg/kg Xanthomycin), basal diets plus 1 g of probiotics B. subtilis product/kg of diets (4 × 108  cfu/kg), basal diets plus 150 mg/kg xylooligosaccharide (35%) and 1 g/kg mannanoligosaccharide (75%), and basal diets plus synbiotics (1 g of probiotics B. subtilis product/kg of diets (4 × 108  cfu/kg), 150 mg/kg xylooligosaccharide (35%) and 1 g/kg mannanoligosaccharide (75%). The results demonstrated that on 21 and 42 days, dietary supplementation of the synbiotics significantly increased daily weight gain (p < 0.05), feed efficiency (p < 0.05), the villus height and villus:crypt ratio in the duodenum, jejunum and ileum (p < 0.05), as well as intestinal mucosa sIgA content (p < 0.05), serum T-SOD activity (p < 0.05) and lysozyme content (p < 0.05), comparing with control group. In conclusion, synbiotics (B. subtilis and xylooligosaccharide and mannanoligosaccharide) is one of the safe and ideal dietary supplementations to increase broilers' growth performance by improving small intestinal morphology, sIgA content and antioxidant capabilities.

Establishment of a novel human esophageal squamous cell carcinoma cell line (ESC-410) and its partial biological characterization.

Esophageal cancer exhibits an uneven geographical distribution strikingly, resulting in focal endemic high-incidence areas in several countries worldwide including China, which might be associated with the environmental and genetic risk factors in those areas. Permanent cancer cell lines are invaluable tools in understanding the biology of cancers and experimental therapeutics. To enrich cell line panel and animal models of human esophageal squamous cell carcinoma (ESCC) from different geographical areas and investigate the environmental and genetic risk factors in the carcinogenesis of ESCC, a novel human esophageal squamous cancer cell line (ESC-410) was established. The cell line grew adherent as a monolayer and maintained stable growth rate with a doubling time of 53 h and distinct epithelial morphological appearance; it was maintained in vitro for 18 months and subcultured for more than 50 passages. Ultrastructural examination revealed large irregular nuclei, desmosome, and tonofilaments; karyotype analysis showed a modal number of chromosomes that ranged from 35 to 73, with a median of 57, and 77% of analyzed cells were hyperdiploidy; reverse transcription polymerase chain reaction (RT-PCR) detected the mRNA expressions of CK8, CK18, and CK19 in the established cells; immunofluorescence assay identified the protein expressions of neurotrophin receptor p75 and integrin α6 (CD49f) in the ESC-410 cell line; xenotransplantation of ESC-410 cells into athymic nude mice subcutaneously induced the formation of solid tumor masses in about 2 weeks. By histopathological examination, heterogeneity of xenograft tumor was observed, as same as that of human primary ESCC. All findings and evidence in this experimental study suggested that this cell line might be a useful model in vitro and in vivo in cellular and molecular studies as well as in testing novel therapies for human ESCC.

Effect of Saccharomyces cerevisiae fermentation product on immune functions of broilers challenged with Eimeria tenella.

Three hundred sixty 1-d-old male Arbor Acres broilers were randomly allotted to 6 groups with a 2x3 factorial arrangement of treatments. Three supplemental levels (0, 0.25, and 0.50%) of Saccharomyces cerevisiae fermentation product (XP) were fed to control and Eimeria tenella-infected broilers. Growth performance and immune response criteria were measured after coccidian infection. Broiler ADG was lowered (P<0.01) by coccidian infection and improved by XP supplementation (P=0.04). Supplementation of XP increased CD3+, CD4+, and CD8+ T-lymphocyte counts (P<0.05) and the ratio CD4+:CD8+ in blood (P=0.06) and spleen (P=0.04) as well as ileum intraepithelial lymphocyte count, cecal tonsil secretory IgA counts, serum lysozyme content (P<0.01), and albumin:globulin ratio (P=0.02). These results suggest that dietary XP supplementation could improve immune function and growth performance in coccidia-infected broilers.

Dietary vitamin A can improve immune function in heat-stressed broilers.

This experiment was undertaken to evaluate the effect of dietary vitamin A on the performance and immune competence of broilers under heat stress (HS). A total of 180 birds, at 22 days of age, were randomly assigned to be reared either at 24°C (thermoneutral, TN, 24°C, constant) or 24°C to 38°C (heat stress, HS, cycling) until the age of 42 days. Birds were then supplemented with vitamin A at 750, 1500, 15 000 IU/kg. Each of the 2 × 3 factorially arranged treatments were replicated in six cages, each containing five birds. Humoral immunity was assessed by intravenous injection of 7% sheep red blood cells (SRBC) followed by evaluation of serum for antibody titers in primary and secondary responses. Cell-mediated immunity was assessed by using a Sephadax stimulation method to recruit abdominal exudate cells (AEC) to evaluate macrophage phagocytic ability. Body weight (BW) and feed conversion were significantly affected by dietary vitamin A (P < 0.05). HS significantly reduced BW, feed intake and feed conversion (P < 0.05). Numbers of AEC, percentage of macrophages in AEC, phagocytic macrophages, internalized opsonized and unopsonized SRBC were increased by dietary vitamin A (P < 0.05). Both primary and secondary antibody responses were characterized by increasing titers of antibody to SRBC by dietary vitamin A when birds were exposed to HS (P < 0.05). Lymphoid organ weights, antibody responses, incidence of macrophages in AEC and phagocytic ability of macrophages were all significantly reduced under HS. These results indicated that HS severely reduced performance and immunocompetence of broilers, whereas the immune response of broilers improved by dietary vitamin A supplementation under HS.

Effects of yeast culture in broiler diets on performance and immunomodulatory functions.

A study was conducted to evaluate the effect of supplemental yeast culture (Diamond V XP Yeast Culture; YC) in broiler diets on performance, digestibility, mucosal development, and immunomodulatory functions. One-day-old Arbor Acres chicks (n = 960) were randomly assigned to 1 of 4 dietary treatments based on corn and soybean meal and containing 0, 2.5, 5.0, and 7.5 g/kg of YC in the diet for 42 d. Each treatment consisted of 12 replicates of 20 broilers each. Nutrient digestibility was determined on d 15 and 35 by total fecal collection. On d 21 and 42, 12 birds per treatment were sacrificed to evaluate gut morphology and secretory IgA. Broilers were vaccinated with Newcastle disease vaccine by eye drop on d 7 and 28 and antibody titer was determined on d 14, 21, 35, and 42. Dietary supplemental YC at 2.5 g/kg improved average daily gain and feed conversion during grower and overall periods (P 0.05) protein retention and energy digestibility. Villus height to crypt depth ratios in the duodenum and jejunum (d 42) and ileum (d 21) were increased (P

Effect of dietary crude protein and energy on gosling growth performance and carcass trait.

The study was undertaken to assess dietary CP and ME concentrations for optimum growth performance and carcass characteristics of goslings. In a 5 x 3 factorial arrangement, 360 one-day-old commercial generation Huoyan goslings were randomly assigned to experimental diets with 10.87, 11.37, 11.87, 12.37, and 12.87 MJ of ME/kg of diet; each contained 15.0, 17.5, and 20.0% CP, respectively, from 0 to 4 wk of age (WOA). Each dietary treatment was replicated 6 times. Body weight and feed consumption were measured, and carcass characteristics were evaluated at 4 WOA. The result showed that birds on a diet with 11.87, 12.37, and 12.87 MJ of ME/kg at 0 to 4 WOA exhibited greater BW gain than those on a diet with 10.87 and 11.37 MJ of ME/kg (P < 0.01), though BW gain was not different among 11.87, 12.37, and 12.87 MJ of ME/kg of diet. Mean BW gain of birds fed 17.5 and 20.0% CP diets was not different (P > 0.05), but they were higher than those on 15.0% dietary CP concentration (P < 0.001). Feed intake was not influenced by dietary ME levels (P > 0.05). Feed intake of birds fed 17.5 and 20.0% CP diets was higher than those of birds on 15.0% CP diets (P < 0.01). Feed conversion ratios of birds fed on 11.87, 12.37, and 12.87 MJ of ME/kg of diet were better than those fed on 10.87 and 11.37 MJ of ME/kg (P < 0.001). Feed conversion ratios of birds fed on 17.5 and 20.0% CP diets were better than those fed on 15.0% CP diets. Moreover, there were no significant interactions between CP and ME on growth performance. There was a direct relationship between dietary ME levels and eviscerated carcass percentage, abdominal fat percentage, and liver relative weight (P < 0.01). Breast and leg meat percentage were influenced by dietary CP concentrations significantly (P < 0.001). Thus, diets with 11.87 MJ of ME/kg and 17.5 to 20.0% CP were used more efficiently from 0 to 4 WOA by Huoyan goslings.

Characterization and expression of plasma and tonoplast membrane aquaporins in primed seed of Brassica napus during germination under stress conditions.

Two aquaporin genes were isolated from a cDNA library of canola (Brassica napus L.). The first aquaporin, BnPIP1 of 1094 bp, encoding a putative polypeptide of 287 amino acids with a predicted molecular mass of 30.4 kDa and a pI of 7.8, belongs to the family of plasma membrane intrinsic protein (PIPs) aquaporins. The B. napus aquaporin showed 85-94% identity to the Arabidopsis thaliana PIPs. ABA priming of seed induced high levels of BnPIP1 transcript which remained after subsequent re-drying of the seed. The second aquaporin, Bny-TIP2 of 1020 bp, encoded a putative polypeptide of 253 amino acids with a predicted molecular mass of 25.8 kDa and a pI of 5.8. Bngamma-TIP2 showed 83-90% identity to gamma-TIP genes from a variety of plant species. Bngamma-TIP2 was expressed only when radicle protrusion occurred in either untreated or primed seeds. Seeds primed with PEG or ABA germinated earlier and showed a higher final percentage of germination than unprimed seed, particularly under salt and osmotic stresses at low temperature. Transcripts of both BnPIP1 and Bngamma-TIP2 genes were present earlier during germination of primed seeds than non-primed seed. From these results, we conclude that BnPIP1 is related to the water transportation required for enzymatic metabolism of storage nutrients at the early stages of canola seed germination whereas Bngamma-TIP2 expression is related to cell growth associated with radicle protrusion. Priming induced the expression of BnPIP1 but had no effect on Bngamma-TIP2.

The detection of thyrotropin-releasing hormone (TRH) and TRH receptor gene expression in Siberian hamster testes.

Thyrotropin-releasing hormone (TRH) from the hypothalamus is the major regulator of TSH synthesis and secretion. Most recently, TRH and TRH receptors (TRH-R), as well as their mRNAs, have been identified in rat testis. To expand our knowledge on the testicular TRH and TRH receptor gene expression in different species, in the present study the mRNA levels of testicular TRH and TRH-R were investigated in Siberian hamsters. To further localize the cellular sites of the gene expression, the animal model was treated with a single injection of ethylene dimethane sulfonate (EDS) (i.p., 80 mg/kg body weight), a compound known as to specifically eliminate testicular Leydig cells. The elimination of Leydig cells induced by EDS treatment was confirmed by histological studies of the testis sections and by serum hormonal analyses, which showed a dramatic reduction of serum testosterone (T) levels and significantly elevated serum LH concentrations. Messenger RNA levels of TRH and TRH-R in the testes were determined by Northern blot analyses quantitated with densitometry scanning. The results showed that specific TRH-R mRNA, 3.8 kb in size, was identified in Siberian hamster testes and the mRNA levels were significantly elevated in the EDS-treated testes compared to the controls (p < 0.01). Testicular TRH mRNA was also detected; however, no significant differences in TRH mRNA levels were found between EDS-treated and control groups. The size of TRH mRNA was characterized as about 1.2 kb in hamster testes, which was smaller than that observed in the rat hypothalamus (1.6 kb) and in the rat testis (2.0 kb). Further studies by RNase H digestion revealed the presence of smaller TRH transcripts in the hamster testes than those in the rat testis. No hybridization signal for TRH mRNA was detected by RNase protection assay, when a rat TRH riboprobe was applied to hamster testis RNA, suggesting the limited homology of TRH gene sequences between these two species. Our results demonstrate that both TRH and TRH-R genes are expressed in Siberian hamster testes, and a significant increase of TRH-R mRNA levels occurs in the Leydig cell eliminated hamster testes. Unlike the rat testicular TRH mRNA mainly detected in Leydig cells, in hamster TRH mRNA could also be detected in other testicular compartment.

An AAV promoter-driven neuropeptide Y gene delivery system using Sendai virosomes for neurons and rat brain.

An adeno-associated virus (AAV)-derived construct (pJDT95npy) containing rat neuropeptide Y (NPY) cDNA inserted downstream of endogenous AAV promoters was used to investigate AAV-driven NPY expression in postmitotic neurons in vitro and in the brain. NPY mRNA was expressed in NT2/N and rat brain primary neuronal cultures after transfection. There was a corresponding increase in the number of neurons staining for NPY-like immunoreactivity and an increase in NPY release during depolarization in the primary cultures. Injections of Sendai-virosome encapsulated pJDT95npy into neocortex increased NPY-like immunoreactivity in neurons but not glia indicating that the latter cell type did not have the translational, post-translational or storage capacity to accumulate the peptide. Injections into the rat hypothalamic para-ventricular nucleus increased body weight and food intake for 21 days, though NPY-like immunoreactivity remained elevated for at least 50 days. These studies demonstrate that AAV-derived constructs may be useful for delivering genes into post-mitotic neurons, and that Sendai virosomes are effective for delivering these constructs in vivo.

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis.

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1-2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4-5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA3, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.

A survey of accessory auricle anomaly. Pedigree analysis of seven cases.

We performed an investigation of 50 accessory auricles on subjects encountered during 1983 through 1985. Fifteen of the families were found to have the same accessory auricular deformity among the probands' family members. From these 15 families, we selected seven pedigrees that were not accompanied by auricular deformities who had filial generations for analysis. Pedigrees 1 through 6 showed autosomal dominant inheritance; two also had irregular dominant properties (pedigrees 4 and 7), and one (pedigree 7) could not be excluded from the possibility of having an X-linked recessive inheritance. During the investigation, there were latent accessory auricles in which the cartilages were seen to be subcutaneous only or protruding slightly. Genetically, the pathogenesis of an accessory auricle should be related to the accessory auricular gene.