Gonadotropin-releasing hormone analog buserelin causes neuronal loss in rat gastrointestinal tract

Gonadotropin-releasing hormone (GnRH) analogs are given to women undergoing in vitro fertilization. Case reports describing the development of chronic intestinal pseudo-obstruction and auto-antibodies against GnRH after such treatment suggest a strong association between intestinal dysfunction and GnRH analogs. No experimental model for studying such a relationship is currently at hand. Our main goal was to investigate possible enteric neurodegeneration and titers of GnRH antibodies in response to repeated administration of the GnRH analog buserelin in rat. Rats were treated for 1–4 sessions with daily subcutaneous injections of buserelin or saline for 5 days, followed by 3 weeks of recovery. Buserelin treatment caused significant loss of submucous and myenteric neurons in the fundus, ileum, and colon. The loss of enteric neurons can, at least partly, be explained by increased apoptosis. No GnRH- or GnRH-receptor-immunoreactive (IR) enteric neurons but numerous luteinizing hormone (LH)-receptor-IR neurons were detected. After buserelin treatment, the relative number of enteric LH-receptor-IR neurons decreased, whereas that of nitric-oxide-synthase-IR neurons increased. No intestinal inflammation or increased levels of circulating interleukins/cytokines were noted in response to buserelin treatment. Serum GnRH antibody titers were undetectable or extremely low in all rats. Thus, repeated administrations of buserelin induce neurodegeneration in rat gastrointestinal tract, possibly by way of LH-receptor hyperactivation. The present findings suggest that enteric neurodegenerative effects of GnRH analog treatment in man can be mimicked in rat. However, in contrast to man, no production of GnRH auto-antibodies has been noted in rat.     

Down-Regulation of eIF4GII by miR-520c-3p Represses Diffuse Large B Cell Lymphoma Development

Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.     

High-Level Production of Human Parathyroid Hormone (hPTH) by Induced Expression inSaccharomyces cerevisiae

Saccharomyces cerevisiaewas used as host for high-level production of intact human parathyroid hormone (hPTH). The yield increased about 30-fold by changing from the constitutive MFα promoter to the inducibleCUP1promoter in the expression cassettes, use of another host strain, and optimization of growth conditions where especially the pH value was crucial. The secreted products consisted mainly of intact hormone, hPTH(1-84). In addition, two C-terminally truncated forms that lacked the four or five last amino acid residues, hPTH(1-80) and hPTH(1-79), were identified. These hPTH forms migrated aberrantly by SDS–PAGE as 14-kDa proteins, while the real masses measured by mass spectrometry on HPLC-purified products were about 9 kDa. Availability of such easily purified truncated forms will be valuable for studies of how the C-terminal residues affect the structure and function of the hormone. Combination of mutations and disruptions of the host genes encoding proteinase A, B, carboxypeptidase Y, and Kex1p or Mkc7p did not influence the C-terminal deletions. The secretion of hPTH could be enhanced by overexpression of the yeast syntaxin geneSSO2, but the total level of the hormone was not improved due to impaired growth.     

Phytochemical analysis of alkaloids from the Icelandic club moss Diphasiastrum alpinum

8S-O-acetylepiclavolonine, a new Lycopodium alkaloid, was isolated from the Icelandic club moss Diphasiastrum alpinum and its structure and stereochemistry was determined using a combination of NMR spectroscopy and mass spectrometry. The previously described alkaloids 5R,8R-O-acetylfawcettiine, 5R,8S-O-acetyllofoline, lycodoline and anhydrolycodoline were found in D. alpinum for the first time together with previously reported lycopodine and clavolonine. The ¹H NMR spectra of 8S-O-acetylepiclavolonine and 5R,8S-O-acetyllofoline, revealed a highly deshielded proton at ca. 12 ppm, which seems to be stabilized by an intramolecular hydrogen bond to the carbonyl oxygen of the 8S-O-acetyl group. The isolated alkaloids were tested toward acetylcholinesterase in vitro without showing significant inhibition.     

Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates

Whole-cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco-efficient route to produce phenolic amines. Here, a novel cell growth-based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from Chromobacterium violaceum (Cv-ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp-SpuC-II and Cv-ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94- and 92-fold increased specific transaminase activity, respectively. Whole-cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp-SpuC-II and Cv-ATA, respectively. Still, amine production was limited by a substantial re-assimilation of the product and formation of the by-products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv-ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re-assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement.     

Autometallographic demonstration of gold in the adrenal gland of rats exposed to sodium aurothiomalate

The presence of gold was investigated in sections of the adrenal glands from rats which had been exposed to intraperitoneal sodium aurothiomalate (32 to 120 mg). Gold was histochemically detected in cortical endocrine cells, chromaffin cells and in fibroblasts and macrophages of both the cortex and medulla. Invisible traces of gold were silver enhanced by autometallography making them readily visible at both the light and electron microscopic levels. The intracellular staining intensity was dose-dependent. In general, the number as well as the staining intensity of individual cells, were highest in the zona glomerulosa and zona reticularis. In gold-containing cells the silver-amplified deposits were present in lysosomes.     

Hypotension and disseminated intravascular coagulation following intralesional bacillus Calmette-Guérin therapy for locally metastatic melanoma

Summary Four patients developed serious hypotension and signs of disseminated intravascular coagulation shortly after a second round of Tice bacillus Calmette-Guérin (BCG) injections into locally recurrent cutaneous melanoma satellite nodules. Each of these patients survived following intensive therapy with isoniazid, pyridoxine, steroids, pressors, antibiotics, and cardio-renal support including, in one case, three acute hemodialyses. Plasma specimens from two of the four patients caused gelation of lysate from the amebocytes ofLimulus polyphemus, indicating the presence of endotoxin or an endotoxin-like substance. In vitro studies on the BCG preparations led us to conclude that this endotoxin activity in the plasma is not the result of direct injection of endotoxin with the BCG preparation, but rather from release of endotoxin from endogenous sources, such as the intestinal tract during a period of relative hypotension following an allergic reaction. Prior immunity appeared to be the consistent factor in the toxic reactions reported herein. Finally, we present recommendations for serial monitoring of these patients and discuss the use of an alternative agent for intralesional therapy.     

The association among herbivory tolerance,ploidy level,and herbivory pressure in <Emphasis Type="Italic">cardamine pratensis</Emphasis>

We tested whether differences in ploidy level and previous exposure to herbivory can affect plant tolerance to herbivory. We conducted a common garden experiment with 12 populations of two ploidy levels of the perennial herb Cardamine pratensis (five populations of tetraploid ssp. pratensis and seven populations of octoploid ssp. paludosa). Earlier studies have shown that attack rates by the main herbivore, the orange tip butterfly Anthocharis cardamines, are lower in populations of octoploids than in populations of tetraploids, and vary among populations. In the common garden experiment, a combination of natural and artificial damage significantly reduced seed and flower production. We measured tolerance based on four plant-performance metrics: survival, growth, seed production and clonal reproduction. For three of these measurements, tolerance of damage did not differ between ploidy levels. For clonal reproduction, the octoploids had a higher tolerance than the tetraploids, although they experience lower herbivore attack rates in natural populations. Populations from sites with high levels of herbivory had higher tolerance, measured by seed production, than populations with low levels of herbivory. We did not detect any significant costs of tolerance. We conclude that high intensity of herbivory has selected for high tolerance measured by seed production in C. pratensis.     

Mouse hallucal metatarsal cross‐sectional geometry in a simulated fine branch niche

Mice raised in experimental habitats containing an artificial network of narrow “arboreal” supports frequently use hallucal grasps during locomotion. Therefore, mice in these experiments can be used to model a rudimentary form of arboreal locomotion in an animal without other morphological specializations for using a fine branch niche. This model would prove useful to better understand the origins of arboreal behaviors in mammals like primates. In this study, we examined if locomotion on these substrates influences the mid‐diaphyseal cross‐sectional geometry of mouse metatarsals. Thirty CD‐1/ICR mice were raised in either arboreal (composed of elevated narrow branches of varying orientation) or terrestrial (flat ramps and walkways that are stratified) habitats from weaning (21 days) to adulthood (≥4 months). After experiments, the hallucal metatarsal (Mt1) and third metatarsal (Mt3) for each individual were isolated and micro‐computed tomography (micro‐CT) scans were obtained to calculate mid‐shaft cross‐sectional area and polar section modulus. Arboreal mice had Mt1s that were significantly more robust. Mt3 cross sections were not significantly different between groups. The arboreal group also exhibited a significantly greater Mt1/Mt3 ratio for both robusticity measures. We conclude that the hallucal metatarsal exhibits significant phenotypic plasticity in response to arboreal treatment due to habitual locomotion that uses a rudimentary hallucal grasp. Our results support the hypothesis that early adaptive stages of fine branch arboreality should be accompanied by a slightly more robust hallux associated with the biomechanical demands of this niche. J. Morphol. 276:759–765, 2015. © 2015 Wiley Periodicals, Inc.