The effects of acute lipopolysaccharide challenge on dairy goat liver metabolism assessed with 1 HNMR metabonomics.

We investigated the mechanisms mediating hepatic metabolic responses to an acute lipopolysaccharide (LPS) challenge in goats. Guanzhong dairy goats (15) were randomly divided into three groups: control (CTL, saline, 0.2 ml/kg BW), lower dose LPS (LPS-L, 20 µg/kg BW) and higher dose LPS (LPS-H, 40 µg/kg BW). All injections were administered intraperitoneally twice with a 24-h interval. Forty-eight hours after the first injection, blood samples were collected to extract plasma for biochemical analysis, and liver tissues were biopsied and stored in liquid nitrogen for metabonomics analysis. We found that plasma levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin increased (p < 0.05) in both LPS-treated groups, whereas plasma triglyceride, cholesterol, very low-density lipoprotein, low-density lipoprotein, high-density lipoprotein, total protein and albumin levels markedly decreased (p < 0.05). The increased activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), levels of tumour necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6 and IL-8 indicated hepatic injury and metabolic dysfunction in some degree. Using proton nuclear magnetic resonance (1 H-NMR) metabonomics and the Chenomx NMR suite database, 69 metabolites were detected and identified. Metabolic differences among the groups were determined with pattern recognition analyses using principal component analysis and supervised projection to latent structures discriminant analysis. Pattern recognition analysis distinguished and clustered the metabolite variables from the three groups, finding nine of 69 metabolites that differed significantly between two of the three groups: six from the LPS-L or LPS-H groups differed from CTL and three differed between LPS-L and LPS-H groups. These altered metabolites were closely connected with glucose, lipid and amino acid metabolic pathways in hepatocytes. Based on an analysis of these metabolites and their relevant pathways, the mechanisms and degree of hepatic injury were deduced. Therefore, the metabolic profile was used effectively to detect characteristic hepatic metabolites, discriminate metabolic changes induced by LPS, clarify the mechanisms for the resulting metabolic dysfunctions and provide efficient information to diagnose liver injury.

ACTH-induced stress in weaned sows impairs LH receptor expression and steroidogenesis capacity in the ovary.

BACKGROUND: Stress has been proved to impair the porcine reproduction soundly. Endocrine disruption, which is closely related to the persistent follicles, is possibly one of the results of stress, although the mechanism is unclear. Since the expression of luteinizing hormone receptor (LHR) in ovarian follicular wall and concentrations of steroid hormone in follicular fluid are related to the development of persistent follicles, this study is designed to evaluate the effect of administered adrenocorticotrophic hormone (ACTH) to weaned pigs on their ovarian steroidogenesis capacity and LHR expression. METHODS: Ten multiparous sows were weaned and randomly divided into two groups (n = 5 each). Sows received 1 IU/kg ACTH (ACTH group) or saline (control group) every 8 h from days 3-9 after jugular vein intubation. Blood samples were collected throughout the experiment, and ovaries were collected after slaughter on day 10. Follicular fluid (FF) was used to determine the steroid hormone concentrations. The ovarian follicle wall was obtained and stored in liquid nitrogen to detect mRNA levels. RESULTS: The plasma cortisol concentration was significantly (P < 0.01) elevated after ACTH injection. The estradiol (E2) and androstenedione (ASD) concentrations in FF were significantly lower (P < 0.05) in the ACTH group than in the control group. The LHR, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 aromatase (P450arom), and cytochrome P450 17a-hydroxylase (P450c17) mRNA levels were significantly (P < 0.05) reduced in the ACTH group. The steroidogenic acute regulatory protein (StAR) level and cytochrome P450 side-chain cleavage (P450scc) was lower in the ACTH group than in the control group, but the difference was not statistically significant (P > 0.05). Immunostaining results revealed 3ß-HSD,P450c17, and LHR expression in theca cells, and P450arom expression in granulosa cells. Immunohistochemical staining showed significant differences in the distribution of 3ß-HSD, P450c17, LHR, and P450arom between the two groups. CONCLUSIONS: These findings indicated that ACTH significantly diminished the LHR expression and steroidogenesis capacity of the ovaries of weaned sows.

Cloning and functional analysis of the promoter of a maize starch synthase III gene (ZmDULL1).

The ZmDULL1 gene encodes a starch synthase and is a determinant of the structure of endosperm starch in maize (Zea mays L.). However, little is known regarding the regulatory mechanism of the ZmDULL1 gene. In this study, we isolated and characterized the ZmDULL1 promoter (PDULL1), which is the 5' flanking region of ZmDULL1 in maize. Sequence analysis showed that several cis-acting elements important for endosperm expression (GCN4_motif and AACA-element) were located within the promoter. A series of PDULL1 deletion derivatives, PDULL1-1-PDULL1-4, from the translation start code (-1676, -1216, -740, and -343) were fused to the ß-glucuronidase (GUS) reporter gene. Each deletion construct was transformed into rice using the Agrobacterium-mediated method, and then GUS activity was measured in transgenic plants. The results showed that PDULL1 was an endosperm-specific promoter. Further analysis showed that the promoter sequence (-343 to -1 base pairs) was sufficient for mediating GUS gene expression in endosperm. These results indicate that the region from -343 to -1 base pairs of PDULL1 is valuable for transgenic rice breeding and genetic engineering studies.

Association between the 8q24 rs6983267 T/G polymorphism and prostate cancer risk: a meta-analysis.

Recent studies have indicated that single nucleotide polymorphisms (SNPs) within the 8q24 region may be a risk factor for prostate cancer (PCa). Here, we performed a meta-analysis to evaluate the association between the 8q24 rs6983267 T/G polymorphism and PCa risk. A systematic literature search was carried out in multiple electronic databases independently by two investigators. Pooled odds ratios (ORs) and 95% confidence intervals for 8q24 rs6983267 T/G and PCa were calculated using a fixed-effect model (the Mantel-Haenszel method). In total, 24 case-control studies from 19 articles were included in our meta-analysis. Our analysis indicated that there is a significant PCa risk associated with the rs6983267 polymorphism in a dominant model (GG vs GT+TT, pooled OR = 1.298, P < 0.001); recessive model (GG+GT vs TT, pooled OR = 1.302, P < 0.001); and homozygote comparison (GG vs TT, pooled OR = 1.494, P < 0.001). Similarly, in a subgroup analysis of European and Asian descent, our results revealed that there are associations between rs6983267 T/G polymorphism and PCa susceptibility with the dominant model (GG vs GT+TT), recessive model (GG+GT vs TT), and homozygote comparison (GG vs TT). To investigate the association between rs6983267 and risk of PCa under different clinical conditions, further analyses were conducted regarding different clinical characteristics including the Gleason score, tumor stage, and PSA level to provide a more comprehensive view of PCa risk and this SNP. Publication bias was assed using the Begg test and the Egger test, and none was detected.

Cloning and prokaryotic expression of the porcine lipasin gene.

Lipasin has recently been demonstrated to be involved in lipid metabolism. In this study, two specific primers were used to amplify the lipasin open reading frame from porcine liver tissue. The polymerase chain reaction product was cloned to a pGEM®-T Easy Vector, digested by SalI and NotI, and sequenced. The lipasin fragment was then cloned to a pET21(b) vector and digested by the same restriction enzyme. The recombinant plasmid was transferred to Escherichia coli (BL21), and the lipasin protein was induced with isopropyl-ß-D-thiogalactopyranoside. The protein obtained was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. A pET-lipasin prokaryotic recombinant expression vector was successfully constructed, and a 25.2-kDa protein was obtained. This study provides a basis for further research on the biological function of porcine lipasin.

Attenuated mRNA expression of lipid metabolism genes in primary hepatocytes following lipopolysaccharide treatment in dairy cows.

The influence of ruminal acidosis on ruminal microbiology and metabolite production has received considerable attention, but little is known regarding the systemic manifestations that arise from ruminal acidosis. Lipopolysaccharide (LPS) is released in the gastrointestinal tract upon ingestion of high-grain or high-fat diets, and it has been implicated in the etiology of multiple energy- and lipid-related metabolic disturbances in ruminants. The liver plays a crucial role in the acute phase response to intruding pathogens. The effect of blood LPS in subacute ruminal acidosis on lipid metabolism in the liver has not been established. In this study, cell cultures were photographed using an inverted microscope. We observed that hepatocytes changed their morphologies from irregular triangle to circular (contraction) shapes; the number of contracted cells increased with the increasing LPS doses. This suggests that LPS can promote cell contraction and take off the wall, ultimately leading to cell apoptosis. With changes in LPS exposure, hepatocyte number also changes. We explored lipid metabolism in the liver using quantitative reverse transcription-polymerase chain reaction to detect the expression of key lipid metabolism enzymes in hepatocytes. We found that Toll-like receptor 4 signaling pathway mediated by LPS could attenuate mRNA expression of fatty acid synthesis genes and increase the expression of fatty acid transport genes in primary hepatocytes following LPS treatment in dairy cows.

Lactoferrin mRNA expression in mouse mammary glands during pregnancy and lactation.

Lactoferrin (Lf) is an iron-binding glycoprotein that is produced by mucosal epithelial cells in mammals. Lf has non-immune natural defense functions and biological functions in addition to and distinct from its role in regulating inflammatory responses. Lf also improved some physiological and immunological parameters. Lf is a biomarker for monitoring medical treatment in inflammatory bowel diseases. Current LF research focuses on iron absorption, antimicrobial activity, and the modulation of iron metabolism during inflammation. No systematic research about Lf expression levels in mouse mammary glands during pregnancy and lactation exists. We investigated Lf mRNA expression levels in mouse mammary glands by collecting samples on days 1, 6, 12, and 18 of pregnancy and lactation (six mice per group). The expression levels of Lf mRNA were measured by semi-quantitative reverse transcription polymerase chain reaction using GAPDH as an internal control. Lf mRNA was not expressed in mammary glands on days 1, 6, and 12 of pregnancy, but it was expressed on day 18 (IOD: integrated optical density; Lf(IOD)/GAPDH(IOD) = 0.46). Lf expression levels were higher during lactation stages than during pregnancy stages, and it stabilized at 0.71-0.73 (Lf(IOD)/GAPDH(IOD)) from day 1 to 12 of lactation; however, the difference was not significant (P > 0.05). At day 18 of lactation, Lf expression began to decline (Lf(IOD)/GAPDH(IOD) = 0.61), but the difference was not significant (P > 0.05). Based on these results, the variation in Lf expression levels during developmental stages may be related to its regulatory role in mouse mammary gland immunity.

Molecular characteristics and cloning of two pepper genes AN2 and UPA20.

The complete coding sequences (CDSs) of "Yunnan Purple Pepper No.1" (Capsicum annuum L.) AN2 and UPA20 genes were amplified using the reverse transcriptase polymerase chain reaction on the basis of the conserved sequence information of some Solanaceae plants and known highly homologous pepper expressed sequence tags. The nucleotide sequence analysis of these 2 genes revealed that pepper AN2 gene encoded a protein of 263 amino acids that has high homology with the AN2-like protein of 4 species: tobacco, tomato, potato, and petunia. The UPA20 gene encoded a protein of 341 amino acids that has high homology with the proteins of 3 species: tobacco, petunia, and tomato. The tissue expression analysis indicated that the pepper AN2 gene was overexpressed in the pericarp and placenta; moderately in stems, flowers, and seeds; and weakly in the roots, leaves, and pericarp. The pepper UPA20 gene was overexpressed in the flowers and seeds; moderately expressed in the roots and stems; and weakly expressed in the leaves and placenta. Our findings might form the basis for further research on these 2 pepper genes.

[Epidemiological characteristics of typhoid fever in Fujian Province, 2011-2022].

Objective: To analyze the incidence trend and epidemiological characteristics of typhoid fever in Fujian Province from 2011 to 2022, and understand the high-incidence population and hotspot areas, and provide evidences to develop more targeted prevention and control measures. Methods: The surveillance data of typhoid fever during 2011-2022 in Fujian Province were obtained from the National Disease Reporting Information System and analyzed with SAS 9.4. The spatial autocorrelation analysis of typhoid fever incidence at county/district levels was performed with ArcGlS 10.8. Results: A total of 5 126 cases of typhoid fever were reported in Fujian Province from 2011 to 2022, with an average annual incidence rate of 1.10/100 000. The average annual incidence rate was 0.96/100 000 from 2011 to 2015, 1.49/100 000 from 2016 to 2019, and 0.81/100 000 from 2020 to 2022. The disease occurred all the year round, with high epidemic season from May to September. A total of 23.59% (1 209/5 126) of the cases occurred at the age of 0-4, and 9.62% (493/5 126) at the age of 5-9. The male to female ratio of the cases was 0.97∶1 (2 524∶2 602) for the whole population, 1.19∶1 (925∶777) for people under 10 years old, 0.75∶1 (1 060∶1 404) for people between 10 and 54 years old, and 1.28∶1 (539∶421) for people over 55 years old. Cases in Ningde City accounted for 30.65% (1 571/5 126) of the total cases. Most hotspots were occurred in Ningde City. Recurrent and clustered cases were found in family members. Conclusions: Typhoid fever was prevalent at a low level in Fujian Province during 2011-2022, indicating that strengthening the prevention and control measures should target key areas and populations. The incidence of typhoid fever in Fujian Province showed spatial aggregation phenomenon, and most cases gathered in Ningde City. Intensive study for the influencing factors of spatial clustering should be conducted.

Molecular cloning and tissue expression analyses of two novel pepper genes: heterotrimeric G protein beta 2 subunit and ArcA1.

We isolated two transcription factor genes, heterotrimeric G protein beta 2 subunit (Gß2) and ArcA1, from pepper (Capsicum annuum). The complete coding sequences were amplified using reversed transcriptase PCR based on conserved sequence information of Solanum lycopersicum and several other plant species. Nucleotide sequence analysis of these two genes revealed that the pepper Gß2 gene encodes a protein of 376 amino acids that belongs to the WD40 superfamily. Tissue expression analysis indicated that this gene is highly expressed in the pericarp, moderately expressed in stem, flower, placenta, and leaves, and weakly expressed in seed. There was no expression in the roots. The ArcA1 gene encodes a protein of 331 amino acids that also belongs to the WD40 superfamily. Tissue expression analysis indicated that the pepper ArcA1 gene is moderately expressed in the pericarp and weakly expressed in seed. There was no expression in root, stem, flower, placenta, or leaves.

Cloning and bioinformatic analysis of full-length novel pepper (Capsicum annuum) genes TAF10 and TAF13.

We isolated two TATA-binding protein-associated factor (TAF) genes, TAF10 and TAF13, from pepper (Capsicum annuum). The complete coding sequences were amplified using reverse transcriptase-PCR on the basis of conserved sequence information of eggplant and several other plant species. Nucleotide sequence analysis of these two genes revealed that the pepper TAF10 gene encodes a protein of 103 amino acids that belongs to the TAF10 superfamily. The pepper TAF10 gene was highly expressed in the pericarp and placenta, moderately expressed in the stems, flowers, seeds and leaves, and weakly expressed in roots. The TAF13 gene was found to encode a protein of 130 amino acids that belongs to the TAF13 superfamily. The TAF13 gene was highly expressed in the stems, flowers and pericarp, moderately expressed in the leaves, placenta and seeds, and weakly expressed in roots.

Molecular cloning, sequence characterization of a novel pepper gene NADP-ICDH and its effect on cytoplasmic male sterility.

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) is an important enzyme involved in energy metabolism. The complete coding sequence of the pepper (Capsicum annuum) NADP-ICDH gene was amplified using a reverse transcriptase PCR based on the conserved sequence information of the tomato and other Solanaceae plants and known highly homologous pepper ESTs. Nucleotide sequence analysis revealed that the pepper NADP-ICDH gene encodes a protein of 415 amino acids that has high homology with the proteins of seven species, Solanum tuberosum (100%), Citrus limon (98%), Daucus carota (98%), Nicotiana tabacum (98%), Vitis vinifera (99%), Arabidopsis thaliana (97%), and Oryza sativa (98%). Tissue expression analysis demonstrated that the pepper NADP-ICDH gene is over expressed in flower, pericarp and seed, moderately in placenta, weakly in stem and leaf, hardly expressed in root. At the abortion stages, activities and expression levels of NADP-ICDH in anthers of a sterile line were strongly reduced, while those in an F(1) hybrid remained normal. Activities and expression levels of NADP-ICDH were too low to maintain balanced energy metabolism in the sterile line, which indicated that stable transcripts of NADP-ICDH are necessary to maintain energy metabolism at a normal level. When the restorer gene was transferred to the cytoplasmic male sterile line, activities and expression level of NADP-ICDH were regulated by the restorer gene and became stable. The restorer gene likely plays an important role in keeping the balance of the energy metabolism within normal levels during microspore development.

IL-8 mRNA expression in the mouse mammary glands during pregnancy and lactation.

Interkeukin-8 (IL-8) is an important inflammatory mediator. It is an angiogenic factor associated with inflammation and carcinogenesis. To date, research on IL-8 has been limited to its role as an indicator of inflammation. There has been no systematic research concerning IL-8 expression levels in the mouse mammary gland during pregnancy and lactation. Mouse mammary gland samples were collected on days 1, 6, 12, 18 of pregnancy and of lactation (6 mice per group). The expression levels of IL-8 mRNA were measured by semi-quantitative RT-PCR, with GAPDH as an internal control. IL-8 mRNA was highly expressed on day 1 of pregnancy in the mouse mammary glands (IL-8(IOD)/GAPDH(IOD) = 1.68), and then suddenly declined; it reached 0.74 and 0.71 on days 6 and 12 of pregnancy. On day 18 of pregnancy, it started to increase (IL-8(IOD)/GAPDH(IOD) = 1.02). However, the expression levels of IL-8 mRNA were not significant during pregnancy. During lactation, IL-8 expression level was lower than during pregnancy, but it stabilized at 0.32-0.41 (IL-8(IOD)/GAPDH(IOD)) from day 1 to day 18 of lactation, although the difference was not significant. We suggest that the changes in IL-8 expression level during development is related to its regulatory role in mouse mammary gland immunity.

[Analysis on repetitive reporting of hepatitis B in Fujian province, 2016-2020].

Objective: To analyze the repetitive reporting of hepatitis B in Fujian province during 2016-2020, and provide evidence for the improvement of hepatitis B surveillance. Methods: The reporting cards from the China Information System for Disease Control and Prevention were collected and divided into repetitive reporting cards and non-repetitive reporting cards from the report cards collected according to the valid ID number on the cards, and the proportion of repetitive report cards and related factors were analyzed by using software SAS 9.4. Results: A total of 314 551 hepatitis B reporting cards were submitted in Fujian from 2016 to 2020, in which 90.93% (286 020/314 551) were included in the analysis. The repetitive reporting cards accounted for 10.48% (29 982/286 020). The annual proportion of the repetitive reporting cards from 2016 to 2020 was between 2.98% and 3.71%, showing an overall increasing trend year by year (Z=2.26, P=0.024). The proportions of the repetitive reporting cards in 1-5 years were 3.17%, 5.40%, 7.74%, 9.27% and 10.48%, respectively, showing an increase trend with year (Z=128.16, P<0.001). The proportions of the repetitive reporting cards in 10 areas of Fujian ranged from 5.44% to 13.48% with significant difference (χ2=2 050.41, P<0.001) and increased with the increase of reported incidence of hepatitis B (Z=26.92, P<0.001). There were significant differences in relationships between repetitive reporting proportion and sex, age and type of the cases between the areas with high incidence and low incidence of hepatitis B. Conclusions: The reported incidence of hepatitis B was seriously affected by the repetitive reporting in Fujian from 2016 to 2020. A cross-year and cross-area surveillance mechanism for hepatitis B should be established and targeted measures should be taken to strengthen the control of the repetitive reporting and improve the surveillance for hepatitis B.

[Incidence of leptospirosis in Fujian province, 2015-2020].

Objective: To analyze the incidence of leptospirosis in Fujian province from 2015 to 2020 and provide the scientific evidences for the risk assessment, prevention and control of leptospirosis. Methods: The incidence data of leptospirosis in Fujian during 2015-2020 were collected from China Information System for Disease Control and Prevention for a descriptive analysis, and software ArcGIS 10.3.1 was used for spatial autocorrelation analysis, and rats were captured in 17 surveillance areas during the same period, and the rat organs were collected for pathogen culture, the level of Leptospira antibody was detected in serum samples of rats, healthy population and the serum samples of patients sent by the hospitals. The infection status of Leptospira in human and rats were analyzed. Results: The incidence of leptospirosis in Fujian showed a downward trend from 2015 to 2020. A total of 176 cases of leptospirosis were reported. There were obvious seasonality and bimodal distribution. The majority of cases were farmers, accounting for 49.43% (87/176). Most cases were aged 30-69 years (85.80%, 151/176). The male to female ratio of the cases was 3.51∶1 (137∶39). Spatial autocorrelation analysis showed that leptospirosis had high or low clustering areas. From 2015 to 2020, the average capture rate of rats in 17 surveillance areas was 6.96% (1 519/21 838), Rattus losea, Rattus flavipectus and Niviventer fulvescens were the main species. The average positive rate of Leptospira antibody in rats was 28.64% (252/880). Java and Autumnalis were the predominant serogroups, accounting for 56.75% (143/252) and 17.46% (44/252), respectively. The average positive rate of Leptospira antibody in healthy population was 16.13% (254/1 575), and Autumnalis and Australis were the predominant serogroups, accounting for 71.65% (182/254). The confirmation rate of leptospirosis in patient serum samples sent by the hospitals was 2.23% (188/8 431), Autumnalis (56.38%, 106/188) and Hebdomadis (19.68%, 37/188) were the major serogroups. Conclusions: The incidence of leptospirosis in Fujian showed a downward trend from 2015 to 2020, there were obvious area clustering and seasonality. The high clustering areas were mainly distributed in northern, western and central Fujian. Java and Autumnalis were the predominant serogroups in rats. The infection rate in healthy population decreased year by year. Autumnalis and Hebdomadis were the main serogroups in population in Fujian.

Carnitine and carnitine orotate affect the expression of the prolactin-releasing peptide gene.

Carnitine is involved in fatty acid metabolism in mammals and is widely used as a nutritional supplement; carnitine orotate is a more absorbable form of carnitine. We investigated the effects of carnitine and carnitine orotate on mouse prolactin-releasing peptide (PrRP) mRNA expression. Twenty-four female mice were randomly divided into four groups of six; control mice were orally drenched with physiological saline solution (250 mg/kg body weight) and treatment mice were orally drenched with carnitine (250 mg/kg) or carnitine orotate (250 or 750 mg/kg), once a day, for 20 days from parturition. The carnitine or carnitine orotate was dissolved in saline solution before administration. The hypothalamus, pituitary and ovary were sampled on day 21 after parturition, and PrRP mRNA levels in these tissues were measured by semi-quantitative PCR, with glyceraldehyde 3-phosphate dehydrogenase as a control. Expression of PrRP in mice treated with carnitine and carnitine orotate was significantly increased in the ovary and significantly reduced in the pituitary gland. Compared with the control, hypothalamus PrRP mRNA increased significantly in the carnitine and low-dose carnitine orotate groups and decreased significantly in the high-dose carnitine orotate group. We conclude that carnitine and carnitine orotate regulate expression of PrRP in the pituitary gland and ovaries.

Prolactin-releasing peptide mRNA expression in mouse medulla remains relatively stable during pregnancy and lactation.

We compared levels of prolactin-releasing peptide (PrRP) mRNA expression in mouse medulla at different stages of pregnancy and lactation. Mouse medulla samples were collected on days 6, 12 and 18 of pregnancy and lactation, respectively (six per group), for mRNA. Expression levels of PrRP mRNA in the medulla were measured by semi-quantitative RT-PCR, with glyceraldehyde 3-phosphate dehydrogenase as a control. PrRP mRNA was highly expressed in mouse medulla oblongata on day 6 of pregnancy (0.53), followed by 0.43 at lactation day 6, and 0.42 at lactation day 12. The expression level of PrRP mRNA on days 12 and 18 of pregnancy and day 18 of lactation shared the same value of 0.36. PrRP mRNA levels during lactation decreased slightly compared with that during pregnancy, but the differences between them were not significant. In summary, PrRP mRNA levels in the medulla oblongata remain relatively stable during pregnancy and lactation. This is evidence that medulla PrRP is not involved in the regulation of prolactin secretion.

Leptin mRNA expression in the rat mammary gland at different activation stages.

Leptin is expressed in various tissues, suggesting that this protein is effective not only at the central nervous system level, but also peripherically. Recent studies have shown leptin production by other tissues, including the placenta, stomach, and mammary tissues, but there is no information available concerning expression levels of leptin in the rat mammary gland at different activation stages. We used semi-quantitative RT-PCR to investigate leptin mRNA expression levels in the rat mammary gland at different activity stages. Rat mammary gland samples were collected from virgin females and on days 6, 12, 18 of pregnancy and of lactation (six rats per group). The expression levels of leptin mRNA were measured by semi-quantitative RT-PCR, with ß-actin as an internal control. Leptin mRNA was highly expressed in virgin rat mammary glands (leptin(IOD)/ß-actin(IOD) = 1.60). It decreased gradually during pregnancy, being lowest at 18 days of pregnancy, when the levels were significantly lower than in virgin mammary tissue. Leptin mRNA increased slightly during lactation, but the difference was not significant. By day 18 of lactation, expression levels of leptin mRNA reached the same values as in virgin mammary tissue (leptin(IOD)/ß-actin(IOD) = 1.65). Based on these results, we suggest that leptin has an important regulation role in rat mammary gland activation.

mRNA abundance and expression of SLC27A, ACC, SCD, FADS, LPIN, INSIG, and PPARGC1 gene isoforms in mouse mammary glands during the lactation cycle.

The functions of distinct isoforms of solute carrier family 27 transporters (SLC27A1-6), acetyl-CoA carboxylase (ACACA, ACACB), stearoyl-CoA desaturase (SCD1-4), fatty acid desaturase (FADS1-3), LPIN (LPIN1-3), insulin-induced gene (INSIG1, 2), and peroxisome proliferator-activated receptor gamma coactivator1 (PPARGC1A, B) were studied in the mouse mammary gland from pregnancy to lactation. The relative mRNA abundance and percent change in real-time PCR were determined. mRNA expression of SLC27A3 and SLC27A4 was 37- and 1.4-fold more upregulated at 12 days of lactation, respectively (P < 0.01). Transcripts of SCD isoforms were the most abundant, accounting for 59% of all genes measured, and PPARGC1 isoforms were the least (0.06% of all genes measured). The mRNA abundance from ACC, FADS and LPIN accounted for 29, 9 and 2.6%, respectively. INSIG1 mRNA expression was 32-fold more upregulated (P < 0.05), while PPARGC1B was 0.18-fold downregulated at 18 days of lactation (P < 0.01). We concluded that mRNA abundance and expression of these isoforms are affected by the stage of lactation.

Selection and use of reference genes in mouse mammary glands.

Obtaining quantitative data concerning gene expression is important for understanding milk synthesis in mammary glands. Quantitative real-time PCR (qRT-PCR) is an efficient tool to calculate gene expression; however, it is necessary to find valid reference genes for normalization of qRT-PCR data. We applied the geNorm software to eight commonly used reference genes to identify the most stable and optimal genes for the mouse mammary gland. Based on this analysis, HPRT, RPL and GAPDH are the most appropriate reference genes for data normalization. We tested the expression of the alpha-lactalbumin and fatty acid synthase genes using these three reference genes, both normalized and non-normalized. The normalized mRNA expression ratio was significantly different from the non-normalized ratio. We recommend the use of these three reference genes for the normalization of qRT-PCR data in gene expression studies of mouse mammary glands.