The establishment of Caenorhabditis elegans germline pattern is controlled by overlapping proximal and distal somatic gonad signals

We investigated the control of proliferation and differentiation in the larval Caenorhabditis elegans hermaphrodite germ line through analysis of glp-1 and lag-2 mutants, cell ablations, and ultrastructural data. After the first several rounds of germ cell division, GLP-1, a receptor of the LIN-12/Notch family, governs germline proliferation. We analyzed the proximal proliferation (Pro) phenotype in glp-1(ar202) and found that initial meiosis was delayed and spatially mispositioned. This is due, at least in part, to a heightened response of the mutant GLP-1 receptor to multiple sources of the somatic ligand LAG-2, including the proximal somatic gonad. We investigated whether proximal LAG-2 affects germline proliferation in the wild type. Our results indicate that (1) LAG-2 is necessary for GLP-1-mediated germline proliferation and prevention of early meiosis, and (2) several distinct anatomical sources of LAG-2 in the larval somatic gonad functionally overlap to promote proliferation and prevent early meiosis. Ultrastructural studies suggest that mitosis is not restricted to areas of direct DTC-germ line contact and that the germ line shares a common cytoplasm in larval stages. We propose that downregulation of the GLP-1 signaling pathway in the proximal germ line at the time of meiotic onset is under tight temporal and spatial control.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

Use of at-line and in-situ near-infrared spectroscopy to monitor biomass in an industrial fed-batch Escherichia coli process

One of the key goals in bioprocess monitoring is to achieve real-time knowledge of conditions within the bioreactor, i.e., in-situ. Near-infrared spectroscopy (NIRS), with its ability to carry out multi-analyte quantification rapidly with little sample presentation, is potentially applicable in this role. In the present study, the application of NIRS to a complex, fed-batch industrial E. coli (RV308/PHKY531) process was investigated. This process undergoes a series of temperature changes and is vigorously agitated and aerated. These conditions can pose added challenges to in-situ NIRS. Using the measurement of a key analyte (biomass) as an illustration, the details of the relationship between the at-line and in-situ use of NIRS are considered from the viewpoint of both theory and practical application. This study shows that NIRS can be used both at-line and in-situ in order to achieve good predictive models for biomass. There are particular challenges imposed by in-situ operation (loss of wavelength regions and noise) which meant the need for signal optimisation studies. This showed that whilst the at-line modelling process may provide some useful information for the in-situ process, there were distinct differences. This study shows that the in-situ use of NIRS in a highly challenging matrix (similar to those encountered in current industrial practice) is possible, and thus extends previous works in the area.     

Development of a large-scale biocalorimeter to monitor and control bioprocesses

Calorimetry has shown real potential at bench-scale for chemical and biochemical processes. The aim of this work was therefore to scale-up the system by adaptation of a standard commercially available 300-L pilot-scale bioreactor. To achieve this, all heat flows entering or leaving the bioreactor were identified and the necessary instrumentation implemented to enable on-line monitoring and dynamic heat balance estimation. Providing that the signals are sufficiently precise, such a heat balance would enable calculation of the heat released or taken up during an operational (bio)process. Two electrical Wattmeters were developed, the first for determination of the power consumption by the stirrer motor and the second for determination of the power released by an internal calibration heater. Experiments were designed to optimize the temperature controller of the bioreactor such that it was sufficiently rapid so as to enable the heat accumulation terms to be neglected. Further calibration experiments were designed to correlate the measured stirring power to frictional heat losses of the stirrer into the reaction mass. This allows the quantitative measurement of all background heat flows and the on-line quantitative calculation of the (bio)process power. Three test fermentations were then performed with B. sphaericus 1593M, a spore-forming bacterium pathogenic to mosquitoes. A first batch culture was performed on a complex medium, to enable optimization of the calorimeter system. A second batch culture, on defined medium containing three carbon sources, was used to show the fast, accurate response of the heat signal and the ability to perfectly monitor the different growth phases associated with growth on mixed substrates, in particular when carbon sources became depleted. A maximum heat output of 1100 W was measured at the end of the log-phase. A fed-batch culture on the same defined medium was then carried out with the feed rate controlled as a function of the calorimeter signal. A maximum heat output of 2250 W was measured at the end of the first log-phase. This work demonstrates that real-time quantitative calorimetry is not only possible at pilot-scale, but could be readily applied at even larger scales. The technique requires simple, readily available devices for determination of the few necessary heat flows, making it a robust, cost-effective technique for process development and routine monitoring and control of production processes.     

Antiproliferative effect of nitric oxide on epidermal growth factor-responsive human neuroblastoma cells

Addition of nitric oxide (NO) donors to NB69 neuroblastoma cells produced a cGMP-independent decrease in cell proliferation, without affecting cell viability or apoptosis. The potency of short half-life NO donors was higher when cell proliferation was stimulated by epidermal growth factor (EGF), as compared with cultures exposed to fetal calf serum (FCS). Immunoprecipitation and western blot analysis of the EGF receptor (EGFR) revealed a significant reduction of its EGF-induced tyrosine phosphorylation in cells treated with the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). When total cell lysates were subjected to western blotting, we observed that DEA-NO also reduced tyrosine phosphorylation in EGF-activated phosphoproteins, but not in those proteins whose tyrosine phosphorylation was evident in the absence of EGF. The effect of NO on EGFR transphosphorylation was concentration-dependent and transient, with a total recovery observed between 1.5 and 3 h after addition of DEA-NO to the cells. When cells were incubated for 15 min with DEA-NO and then washed, the EGFR transphosphorylation returned to control levels immediately, indicating that the interaction of NO with the receptor molecule was fully reversible. NB69 cells expressed both the neuronal and the inducible isoforms of NO synthase (NOS) when cultured in the presence of FCS; under this condition, the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester, produced a small but significant increase in cell proliferation. The results suggest that NO is an endogenous antimitotic agent and that its interaction with EGFR contributes to cytostasis in NB69 cells.     

Establishment and characterization of a cell-line originated from human mucinous cystadenocarcinoma of the ovary

We recently established a cell line (designated 371M) derived from an ovarian mucinous cystadenocarcinoma. The tumor cells were obtained from the ascitic fluid of a 54-year-old Japanese woman while she was undergoing surgery. Adjuvant chemotherapy (combined paclitaxel and carboplatin) was administered, but was ineffective, and she died about 4 months after surgery. The 371M cells continuously propagated in vitro over a period of about 50 months and, to date, have undergone over 100 passages. They proliferated in a monolayered sheet with doubling times of 84 h and 37 h in the 10th and 34th passages, respectively. When transplanted into nude mice, the tumor histopathologically resembled the structure of the original tumor. The 371M cells secreted high levels of CA125 and CA19-9 into the culture medium. There were several abnormal chromosomes in all karyotypes selected at random. Sensitivity of 371M cells to a variety of anti-cancer drugs was examined by in vitro MTT assay, and the results suggested that CPT-11 and CDDP were more effective against 371M cells than other anti-cancer agents.     

Establishment and characterization of a lymphoepithelial-like carcinoma cell line (HUUCLEC) derived from the human uterine cervix

A cell line designated HUUCLEC was established from a human uterine cervical lymphoepithelial carcinoma obtained from a 61-year-old Japanese woman. The cell line has grown slowly without interruption and serial passages were successively carried out 60 times within 3 years. The cultured cells were spindle or round in shape, showing anaplastic and pleomorphic features, a pavement cell arrangement and multilayering without contact inhibition. The population doubling time of the HUUCLEC line was 72 hours while the chromosomal number varied widely and showed aneuploidy. The modal chromosomal number was stable at the triploid range and marker chromosomes were present; the Ebstein-Barr virus was absent in the cultured cells.