Current state and perspectives on erythropoietin production

Erythropoietin is a major regulator of erythropoiesis which maintains the body's red blood cell mass and tissue oxygenation at an optimum level. Recombinant human erythropoietin (rhEPO), which is a widely used therapeutic agent for the treatment of anemia and which represents one of the largest biopharmaceuticals markets, is produced from recombinant Chinese hamster ovary cells. rhEPO is a glycoprotein with complex glycan structure, which is responsible for its therapeutic efficacy, including the in vivo activity and half-life. In order to obtain an optimal and consistent glycoform profile of rhEPO and concurrently maintain a high production yield, various approaches in drug development and cell culture technology have been attempted. Recent advances in rhEPO production are classified into three types: the development of improved rhEPO molecules by protein engineering; improvement of production host cells by genetic engineering; and culture condition optimization by fine control of the production mode/system, process parameters, and culture media. In this review, we focus on rhEPO production strategies as they have progressed thus far. Furthermore, the current status of the market and outlook on rhEPO and its derivatives are discussed.     

Green fluorescent protein-tagged Edwardsiella tarda reveals portal of entry in fish

The application of green fluorescent protein (GFP) to identify the portal(s) of entry of bacterial pathogens in animal hosts was studied using the fish pathogen Edwardsiella tarda and blue gourami, Trichogaster trichopterus. An immersion challenge model was utilized to mimic natural infection conditions in fish. Gastrointestinal tract, gills and the body surface of fish were found to be the sites of entry of virulent E. tarda (PPD130/91) by histological and infection kinetics studies. On the other hand, avirulent E. tarda (PPD125/87) was mainly found in the gastrointestinal tract, and the bacterial population in tissue declined over a period of 7 days.     

Molecular analysis of the SCARECROW gene in maize reveals a common basis for radial patterning in diverse meristems

Maize and Arabidopsis root apical meristems differ in several aspects of their radial organization and ontogeny. Despite the large evolutionary distance and differences in root radial patterning, analysis of the putative maize ortholog of the Arabidopsis patterning gene SCARECROW (SCR) revealed expression localized to the endodermis, which is similar to its expression in Arabidopsis. Expression in maize extends through the quiescent center, a population of mitotically inactive cells formerly thought to be undifferentiated and to lack radial pattern information. Zea mays SCARECROW (ZmSCR), the putative maize SCR ortholog, was used as a molecular marker to investigate radial patterning during regeneration of the root tip after either whole or partial excision. Analysis of the dynamic expression pattern of ZmSCR as well as other markers indicates the involvement of positional information as a primary determinant in regeneration of the root radial pattern.     

Cryopreservation of <Emphasis Type="Italic">Panax ginseng</Emphasis> Adventitious Roots

We tested desiccation and/or vitrification procedures to cryopreserve the adventitious roots of Panax ginseng, the source of commercially produced ginsenosides. When only desiccation was applied, the post-freeze survival of 3- to 4-mm root tips was <14% regardless of the composition of the preculture medium or the explant origin. Callus formation was frequently observed after cryopreservation. In contrast, 90% survival and 32.5% root formation efficiency were achieved after cryopreservation when a vitrification protocol was followed. Adventitious root cultures in flasks and bioreactors were reestablished from root tips cryopreserved by vitrification. A prolonged lag-phase and lower biomass production were recorded in post-freeze-regenerated cultures compared with control roots that were subcultured four times in flasks. However, biomass accumulations did not differ between control and regenerated roots at the end of the sixth subculturing period. After 40 days of culture in bioreactors, a mean value of 12.5 g dw L⁻¹ was recorded for post-freeze-regenerated cultures versus 9.1 g dw L⁻¹ for the control roots. Production of triol and diol ginsenosides in our bioreactor cultures also was enhanced after cryopreservation, by 41.0% and 89.8%, respectively. These results suggest that the vitrification method is successful for cryopreservation of P. ginseng adventitious roots.     

Optimization of culture medium for cloned bovine embryos and its influence on pregnancy and delivery outcome

This study was conducted to establish an effective culture system for supporting in vitro development of cloned bovine embryos and to evaluate whether improved development in the optimal culture system could contribute to enhancing pregnancy and delivery outcomes after transfer. Enucleated oocytes at the metaphase II stage were reconstructed with serum-starved ear fibroblasts and cloned embryos were subsequently cultured for 168 h in vitro. In Experiment 1, cloned embryos were cultured in either modified Charles Rosenkrans 2 amino acid medium (mCR2aa) or modified synthetic oviduct fluid medium (mSOF). More (P < 0.05) 2-cell embryos (78% versus 92%), morulae (51% versus 69%) and blastocysts (2% versus 39%) were obtained after culture in mSOF than after culture in mCR2aa. In Experiment 2, cloned embryos were successively cultured in mSOF supplemented with various macromolecules during different periods of culture. A successive culture of oocytes in BSA-containing medium for 72 h and then in FBS-containing medium for the next 96 h yielded a higher rate of blastocyst formation (49% versus 25-36%) than other combinations (BSA to BSA or PVA to PVA, BSA or FBS). This macromolecule supplementation also significantly increased the number of total blastomeres (117.3 cells/blastocyst) and inner cell mass cells (ICM, 49.7 cells/blastocyst), and the ratio of ICM cells to trophoblast cells (TB, 0.98). In Experiment 3, a total of 85 blastocysts obtained from each 2-step culture were transferred individually to recipient cows at the end of the culture period and 32 pregnancies (38%) were diagnosed on Day 60 after transfer. However, no (P > 0.05) significant differences due to culture were apparent in the pregnancy outcome. Although six calves were produced using the 2-step culture regime of either BSA-BSA or PVA-FBS, no calves were produced using the successive culture of BSA then FBS, which optimized preimplantation development. In conclusion, mSOF has more potential to support the development of clone embryos than mCR2aa, and successive supplementation of BSA and FBS to mSOF further promotes blastocyst formation. However, enhanced development in vitro might not directly contribute to improving pregnancy outcomes.     

Antiproliferative function of glia maturation factor beta.

Recombinant human glia maturation factor beta (GMF-beta) reversibly inhibits the proliferation of neoplastic cells in culture by arresting the cells in the G0/G1 phase. This phenomenon is not target-cell specific, as neural and nonneural cells are equally inhibited. When tested simultaneously, GMF-beta suppresses the mitogenic effect of acidic fibroblasts growth factor (aFGF), but the two are synergistic in promoting the morphologic differentiation of cultured astrocytes. GMF-beta also counteracts the growth-stimulating effect of pituitary extract and cholera toxin on Schwann cells. The results underscore the regulatory role of GMF-beta and its intricate interaction with the mitogenic growth factors.     

Global gene expression analysis to identify molecular markers of uterine receptivity and embryo implantation

Infertility and spontaneous pregnancy losses are an enduring problem to women's health. The establishment of pregnancy depends on successful implantation, where a complex series of interactions occurs between the heterogeneous cell types of the uterus and blastocyst. Although a number of genes are implicated in embryo-uterine interactions during implantation, genetic evidence suggests that only a small number of them are critical to this process. To obtain a global view and identify novel pathways of implantation, we used a dual screening strategy to analyze the expression of nearly 10,000 mouse genes by microarray analysis. Comparison of implantation and interimplantation sites by a conservative statistical approach revealed 36 up-regulated genes and 27 down-regulated genes at the implantation site. We also compared the uterine gene expression profile of progesterone-treated, delayed implanting mice to that of mice in which delayed implantation was terminated by estrogen. The results show up-regulation of 128 genes and down-regulation of 101 genes after termination of the delayed implantation. A combined analysis of these experiments showed specific up-regulation of 27 genes both at the implantation site and during uterine activation, representing a broad diversity of molecular functions. In contrast, the majority of genes that were decreased in the combined analysis were related to host immunity or the immune response, suggesting the importance of these genes in regulating the uterine environment for the implanting blastocyst. Collectively, we identified genes with recognized roles in implantation, genes with potential roles in this process, and genes whose functions have yet to be defined in this event. The identification of unique genetic markers for the onset of implantation signifies that genome-wide analysis coupled with functional assays is a promising approach to resolve the molecular pathways required for successful implantation.     

Low molecular weight polyethylenimines linked by beta-cyclodextrin for gene transfer into the nervous system

BACKGROUND: Polyethylenimines (PEIs) with high molecular weights are effective nonviral gene delivery vectors. However, the in vivo use of these PEIs can be hampered by their cellular toxicity. In the present study we developed and tested a new PEI polymer synthesized by linking less toxic, low molecular weight (MW) PEIs with a commonly used, biocompatible drug carrier, beta-cyclodextrin (CyD). METHODS AND RESULTS: The terminal CyD hydroxyl groups were activated by 1,1'-carbonyldiimidazole. Each activated CyD then linked two branched PEI molecules with MW of 600 Da to form a CyD-containing polymer with MW of 61 kDa, in which CyD served as a part of the backbone. The PEI-CyD polymer developed was soluble in water and biodegradable. In cell viability assays with sensitive neurons, the polymer performed similarly to low-MW PEIs and displayed much lower cellular cytotoxicity compared to PEI 25 kDa. The gene delivery efficiency of the polymer was comparable to, and at higher polymer/DNA ratios even higher than, that offered by PEI 25 kDa in neural cells. Attractively, intrathecal injection of plasmid DNA complexed by the polymer into the rat spinal cord provided levels of gene expression close to that offered by PEI 25 kDa. CONCLUSIONS: The polymer reported in the current study displayed improved biocompatibility over non-degradable PEI 25 kDa and mediated gene transfection in cultured neurons and in the central nervous system effectively. The new polymer would be worth exploring further as an in vivo delivery system of therapeutic genetic materials for gene therapy of neurological disorders.     

An Imported Case of Cystic Echinococcosis in the Liver

A 25-year-old Uzbek male presented with right upper abdominal pain for 20 days. On radiologic studies, a huge cystic mass was noticed in the right liver which was suspected as parasitic. The patient received right hepatic segmentectomy (segment 7), and the surgically resected mass was confirmed as cystic echinococcosis (CE), measuring 10.5 cm in its diameter. The inner surface of the cyst was bile-stained. The patient was discharged on the 8th hospital day, and was rechecked 6 months after the surgical intervention without any evidence of recurrence. The present report describes findings of an imported case of CE which represented ultrasound images of the ''ball of wool''.