Dual role of osteoblastic proenkephalin derived peptides in skeletal tissues

Proenkephalin encodes a group of small peptides with opiate-like activity, the endogenous opioids, known to function as neurohormones, neuromodulators, and neurotransmitters. Recently, we have demonstrated that in addition to its abundance in fetal brain tissue, proenkephalin is highly expressed in nondifferentiated mesodermal cells of developing fetuses. We identified the skeletal tissues, bone, and cartilage as major sites of proenkephalin expression. To examine the possibility that proenkephalin is involved in bone development we have studied the expression of this gene in bone-derived cells, its modulation by bone active hormones, and the effects of enkephalin-derived peptides on osteoblastic phenotype. Our studies revealed that osteoblastic cells synthesize high levels of proenkephalin mRNA which are translated, and the derived peptides are secreted. Reciprocal interrelationships between osteoblast maturation and proenkephalin expression were established. These results together with our observations demonstrating inhibitory effects of proenkephalin-derived peptides on osteoblastic alkaline phosphatase activity, strongly support the notion that proenkephalin is involved in bone development. A different direction of research by other investigators has established the capability of the opioid system in the periphery to participate in the control of pain. On the basis of these two lines of observation, we would like to present the following hypothesis: The potential of embryonic skeletal tissue to synthesize proenkephalin-derived peptides is retained in the adult in small defined undifferentiated cell populations. This potential is realized in certain situations requiring rapid growth, such as remodeling or fracture repair. We suggest that in these processes, similarly to the situation in the embryo, the undifferentiated dividing cells produce the endogenous opioids. In the adult these peptides may have a dual function, namely participating in the control of tissue regeneration and in the control of pain. © 1994 Wiley-Liss, Inc.     

Expression patterns and association analysis of the porcine DHX58 gene

RIG-1 signalling is responsible for the detection of cytoplasmic viral RNA molecules. DEXH (Asp-Glu-X-His) box polypeptide 58 (encoded by DHX58) is a negative regulator of the RIG-1 signalling pathway. In human, the DHX58 gene can be upregulated and can inhibit the RIG-1 signalling pathway during viral infection. In this study, porcine DHX58 gene expression patterns were studied. According to our results, the porcine DHX58 gene was upregulated not only by the stimulation of Poly I:C but also by the stimulation of 1ipopolysaccharides (LPS). One polymorphism (g.4919G>C), detected in the ninth intron, was significantly associated with some blood parameters including the red cell distribution width of 1-day-old pigs and white blood cell counts, lymphocyte absolute counts, and platelet distribution width of 17-day-old pigs (P < 0.05). Moreover, the individuals with the genotype GG have a significantly higher mean white blood cell count than individuals with genotype CC or GC (P < 0.05). Our study indicates that DHX58 is an important gene that is associated with the immune response in swine.     

5S ribosomal gene clusters in wheat: pulsed field gel electrophoresis reveals a high degree of polymorphism

Summary The long-range structure of 5S rRNA gene clusters has been investigated in wheat (Triticum aestivum L.) by means of pulsed field gel electrophoresis. Using aneuploid stocks, 5S rRNA gene clusters were assigned to sites on chromosomes 1B, 1D, 513 and 5D. Cluster sizes were evaluated and the copy number of 5S DNA repeats was estimated at 4700-5200 copies for the short repeating unit (410 bp) and about 3100 copies for the long repeat (500 bp) per haploid genome. A comparison of wheat cultivars revealed extremely high levels of polymorphism in the 5S rRNA gene clusters. With one restriction enzyme digest all varieties tested gave unique banding patterns and, on a per fragment basis, 21-fold more polymorphism was detected among cultivars for 5S DNA compared to standard restriction fragment length polymorphisms (RFLPs) detected with single copy clones. Experiments with aneuploid stocks suggest that the 5S rRNA gene clusters at several chromosomal sites contribute to this polymorphism. A number of previous reports have shown that wheat cultivars are not easily distinguished by isozymes or RFLPs. The high level of variation detected in 5S rRNA gene clusters therefore offers the possibility of a sensitive fingerprinting method for wheat. 5S DNA and other macro-satellite sequences may also serve as hypervariable Mendelian markers for genetic and breeding experiments in wheat.     

Long-term effects of dietary isoflavones on uterine gene expression profiles

Isoflavones (ISOs) are bioactive food ingredients of the traditional East Asian diet and currently discussed as alternatives to classical hormone replacement therapies and for reducing the prevalence of hormone-dependent cancers. Although there are many studies on ISOs, not much is known about their long-term effects.Therefore, we performed an animal experiment analyzing the effects of three different diets: a phytoestrogen-free diet, a diet supplemented with genistein (700 μg/g diet) and an ISO-high diet (232 μg daidzein and 240 μg genistein/g) at two distinct time points, juvenile (21 days) and adult (97 days). Exposure started prior to mating of the parents and throughout the life of the offspring.We observed a stronger increase of uterine wet weights in juvenile offspring with genistein exposure (1018 ± 350 mg/kg BW) than with ISO-high diet (497 ± 133 mg/kg BW). Whereas the expression of proliferation related genes (PCNA; Ki67; IGF-1; IGF-1R), analyzed by real-time-qPCR and Western blot, were significantly down-regulated in juvenile animals exposed to genistein. Additionally, genistein exposure led to estrogenic responses, observed upon increase of complement C3 and decrease of estrogen receptors gene expressions, while the exposure to ISO-high diet did not show these effects.In conclusion, both the time point on which phytoestrogen exposure starts together with the composition of the ingested phytoestrogen containing diet are of great importance for the biological response of the offspring.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Molecular changes associated with tuberisation in Solanum tuberosum. Differential expression of α-tubulin isotypes

Changes in gene expression that occur in the stolon tips of potato ( Solanum tuberosum L.) cv. Record during tuberisation were investigated. Protein extracts from stolon tips at various stages in the tuberisation process were analysed by two-dimensional gel electrophoresis. A number of quantitative and qualitative changes in polypeptide composition accompanied the very early stages of tuberisation. In vitro translation of RNA extracted from stolon tips also revealed quantitative and qualitative changes associated with tuberisation. Immunoblotting of protein extracts with monoclonal antibodies raised against α- and β-tubulin showed quantitative changes in the relative level of β-tubulin, but not α-tubulin, as the stolon tips tuberised. Changes in the pattern of α-tubulin isotype expression were shown to occur at early stages in the tuberisation process.     

Low night temperatures have a differential effect on the diurnal cycling of gene expression in cold–sensitive and tolerant tomatoes*

Abstract. Comparisons were made between the changes in mRNA levels induced by low night temperatures in the cold–sensitive tomato and two altitudinal ecotypes of the wild species L. hirsutum. Changes in mRNA levels were detected by resolving in vitro translation products of poly(A)⁺ RNA by 2-D PAGE. The treatment was applied by first growing plants in a thermoperiod of 25/18°C and then switching to 25/6°C. All tomatoes displayed a diurnal cycling in which a set of mRNAs accumulated at the end of the 18°C nights, whereas another accumulated at the end of the 25°C days. The accumulation of night specific mRNAs was inhibited by 6°C nights in the cold sensitive tomatoes while that of the tolerant one was only marginally affected. All tomatoes showed a similar reduction in the apparent turnover rate of the day specific mRNAs during the 6°C nights. Finally, low night temperatures induced the accumulation of six to eight mRNAs in all genotypes. This number increased by 15 in L. esculentum after the seventh night and are likely involved in stress response rather than acclimation/tolerance. The tomato is proposed as a genetic model to discriminate genes involved in acclimation/tolerance from those involved in stress response.     

Regressive evolution: ontogeny and genetics of cavefish eye rudimentation

Two hypotheses exist to explain ontogenetic eye reduction in Astyanax cave fish: first, after lens induction by the primordial eye cup, the lens plays the role of a central regulator of eye and retina regression or, second, the retina itself is an independent unit of eye development. A comparative study of five blind cave fish populations and their surface sister form was performed to investigate the differences of ontogenetic eye regression between the cave populations during different stages of development. The study revealed that, in addition to the initial formation of smaller primordia, eye regression is also caused during later ontogeny by different relative growth and specific histological characteristics. Whereas the cave fish lens never properly differentiates, the regressive process of the retina is transitorily interrupted by ongoing differentiation. In the newly-discovered Molino cave population, even visual cells with well-organized outer segments develop, which are secondarily reduced at a later ontogenetic stage. This result shows that the retina and lens are independent developmental units within the eye ball. Presumably, the genetic systems responsible for both show independent inheritance, which is also corroborated by hybrids of F ₂-crosses between the cave and surface fish, in which lens and retina development do not correlate. During ontogeny, the eye size differs between the cave populations. In Pachón cave fish, the relatively large eye size correlates with an ancient introgression from a surface population, which may have delayed eye regression.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 287–296.