Understanding the role of thyroid hormone in Sertoli cell development: a mechanistic hypothesis

More than a decade of research has shown that Sertoli cell proliferation is regulated by thyroid hormone. Neonatal hypothyroidism lengthens the period of Sertoli cell proliferation, leading to increases in Sertoli cell number, testis weight, and daily sperm production (DSP) when euthyroidism is re-established. In contrast, the neonatal Sertoli cell proliferative period is shortened under hyperthyroid conditions, but the mechanism by which thyroid hormone is able to negatively regulate Sertoli cell proliferation has been unclear. Recent progress in the understanding of the cell cycle has provided the opportunity to dissect the molecular targets responsible for thyroid-hormone-mediated effects on Sertoli cell proliferation. In this review, we discuss recent results indicating a critical role for the cyclin-dependent kinase inhibitors (CDKI) p27^Kip1 and p21^Cip1 in establishing Sertoli cell number, testis weight, and DSP, and the ability of thyroid hormone to modulate these CDKIs. Based on these recent results, we propose a working hypothesis for the way in which thyroid hormone regulates the withdrawal of the cell cycle by controlling CDKI degradation. Finally, although Sertoli cells have been shown to have two biologically active thyroid hormone receptor (TR) isoforms, TRα1 and TRβ1, experiments with transgenic mice lacking TRα or TRβ illustrate that only one TR mediates thyroid hormone effects in neonatal Sertoli cells. Although significant gaps in our knowledge still remain, advances have been made toward appreciation of the molecular sequence of events that occur when thyroid hormone stimulates Sertoli cell maturation. We gratefully acknowledge the support of this work by the NIH, USDA, the University of Illinois, the Lalor Foundation, and the Thanis A. Field Endowment at the University of Illinois. D.R. Holsberger was supported by postdoctoral fellowships from the Lalor Foundation and Reproductive Biology Research Training Program (NIH grant T32 HD07028), University of Illinois at Urbana–Champaign.     

Regulation of progesterone during follicular development by FSH and LH in sheep

Progesterone (P4) can participate in the development of female mammalian antral follicles through nuclear receptor (PGR). In this experiment, the differences of P4 synthesis and PGR expression in different developmental stages of sheep antral follicles (large > 5mm, medium 2-5mm, small < 2mm) were detected by enzyme-linked immunosorbent assay, immunohistochemistry, qRT-PCR and Western blotting. Secondly, sheep follicular granulosa cells were cultured in vitro. The effects of different concentrations of FSH and LH on P4 synthesis and PGR expression were studied. The results showed that acute steroid regulatory protein (StAR), cholesterol side chain lyase (P450scc) and 3β Hydroxysteroid dehydrogenase (3β-HSD) and PGR were expressed in antral follicles, and with the development of antral follicles in sheep, StAR, P450scc and the expression of 3β-HSD and PGR increased significantly. In vitro experiments showed that FSH and LH alone or together treatment could regulate P4 secretion and PGR expression in sheep follicular granulosa cells to varying degrees, hint P4 and PGR by FSH and LH, and LH was the main factor. Our results supplement the effects of FSH and LH on the regulation of P4 synthesis during follicular development, which provides new data for further study of steroid synthesis and function in follicular development.     

A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of ~1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.     

血必净注射液联合比阿培南对重症肺炎患者肺功能的影响

摘要 目的：研究血必净注射液联合比阿培南对重症肺炎患者肺功能的影响。方法：选择2017年1月～2019年12月我院的103例重症肺炎患者,随机分为两组。对照组静脉滴注比阿培南0.3 g ,每 8 h给药1次;观察组联合静脉滴注血必净50 mL,每天两次。检测两组的炎症因子：白介素-1（Interleukin-1, IL-1）、肿瘤坏死因子-α（Tumor necrosis factor-α, TNF-α）和IL-6水平,应激激素：皮质醇（Cortisol, Cor）、人血管紧张素Ⅰ（Human angiotensin I, AngⅠ）、去甲肾上腺素（Noradrenaline, NE）和人血管紧张素Ⅱ（Human angiotensinⅡ, AngⅡ）水平,肺功能：第一秒最大呼气量（Maximum expiratory volume in the first second, FEV₁）、FEV₁%pred、用力肺活量（Forced vital capacity, FVC）。结果：观察组的有效率明显高于对照组（P<0.05）。治疗后,两组的血清IL-1、TNF-α、IL-6、Cor、AngⅠ、NE和AngⅡ水平水平均明显降低,FEV₁、FEV₁%pred和FEV₁/FVC明显升高（P<0.05）,且观察组的上述指标明显优于对照组（P<0.05）。结论：血必净注射液联合比阿培南对重症肺炎有显著的疗效,不但能明显改善肺功能,还能有效抑制患者的应激反应和炎症反应,值得推广。     

Efficient preparation of glycoprotein hormones lacking an alpha-subunit oligosaccharide

The oligosaccharide on alpha-subunit loop 2 (alpha 2) is needed for full glycoprotein hormone efficacy. Efforts to prepare glycoprotein hormone antagonists usually involve removing the alpha 2 oligosaccharide and are hampered by its requirement for efficient heterodimer secretion from mammalian cells. Here we show that hormones lacking this oligosaccharide can be produced by treating them at low pH to dissociate the heterodimer and permitting the subunits to re-associate in the presence of peptide N-glycosidase F (PNGase F). Re-assembly of human choriogonadotropin, human follitropin, and bovine lutropin occurred rapidly and efficiently following removal of the alpha 2 oligosaccharide by PNGase F. Consequently, virtually all heterodimers formed in the presence of this enzyme lacked this oligosaccharide. These findings support the notion that heterodimer assembly in vitro occurs by a threading mechanism that is impeded by the presence of the alpha 2 oligosaccharide. This procedure should facilitate the study of glycoprotein hormone structure and function.     

Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.     

Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells

We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells.