High concentrations of horse serum inhibit growth of corn stunt spiroplasma.

Corn stunt spiroplasma (CSS) grew faster and achieved higher titers in liquid or agar medium containing 5 or 10 percent horse serum than it did in medium containing 20 percent horse serum. When growth in liquid medium was initiated with a small inoculum, CSS achieved excellent growth in the presence of 5 percent serum but did not grow in medium containing 0 or 20 percent serum. Addition of arginine to liquid or agar medium supplemented with 20 percent serum stimulated CSS growth, but addition to that containing 5 percent serum did not.     

Infection of strawberry flowers by Botrytis cinerea and its relevance to grey mould development

Corn stunt spiroplasma (CSS) multiplied in all injected Dulbulus maidis, reaching titres of over 1 × 10⁶ colony forming units (cfu)/insect and 1 × 10⁴ cfu/salivary gland of each insect. Spiroplasmas could be isolated from the haemolymph and from the salivary glands 1 h after injection and at any time subsequently. Insect extract at a concentration greater than the equivalent of 0.1 insects/ml was inhibitory to the growth of CSS in cultures. Helices could be seen in the haemolymph at any time after injection. However, distorted or partially deformed cells and small aggregates were not present until 2–3 wk after injection. The salivary gland cells of injected insects contained membrane-bound ‘pockets’ or ‘colonies’ packed with pleomorphic organisms, which included some filamentous forms. Intracellular colonies were always on the periphery of cells and were easily detectable by fluorescent microscopy. Both pleomorphic and filamentous forms were also seen intercellularly in the salivary glands. Following injection, transmission of CSS to maize and to sterile feeding solution were compared using 1 day feeding periods. A proportion of injected leafhoppers began to transmit to maize by the third day following injection (5%) and reached a maximum of 72% by day 14. By day 9 , 82% of the population had transmitted at least once to plants and by day 12 , 100% had transmitted. Similar insects transmitted through membranes to sterile feeding solution on day 4 (3%) reaching a maximum of 62% by day 14.     

Corn Stunt Spiroplasma in the Leafhopper Euscelidius variegatus

Corn stunt spiroplasma (CSS) multiplied in all leafhoppers Euscelidius variegatus injected with a culture of CSS, reaching titres of over 1x10⁶ colony forming units (cfu) per insect and 2x10⁴ cfu per salivary gland of each insect. CSS could be isolated from the haemolymph and the salivary glands at any time after injection. The growth of CSS in culture was inhibited by insect extract at concentrations greater than the equivalent of 0.1 insect/ml. Transmission of CSS to sterile feeding solution and to broad beans were compared using 24 h feeding periods. A porportion of 1.7 % of injected leafhoppers began to transmit to sterile feeding solution through membranes by the 4th day after injection, and reached a maximum of 30 % by day 14. Similar insects started transmitting to broad bean plants on day 12 (2 %), reaching a maximum of 7.5 % by day 14. The number of spiroplasmas transmitted by each insect to sterile feeding solution increased from 3, cfu on day 4 to a maximum of 80 cfu by day 14. Helices were seen in the haemolymph at any time after injection. However, partially deformed cells were not present until the 1st week and clumps of 3–4 cells and small aggregates until the 3rd week after injection. The salivary glands of injected insects contained membrane-bound “pockets” or “colonies” packed with pleomorphic, filamentous (helical and non-helical) cells and aggregates. Intracellular colonies were always at the periphery of the acini and were easily detectable by fluorescence microscopy after staining with a DNA-binding fluorescent stain. Pleomorphic and filamentous cells were also seen intercellularly in the salivary glands.     

Surface components in adhesion of group a streptococci to pharyngeal epithelial cells

An antiserum was prepared in rabbits against an isolate of corn stunt spiroplasma (CSS; I-747). The immunoglobulin of the antiserum was purified and conjugated with alkaline phosphatase by standard procedures and used in the enzyme-linked immunosorbent assay (ELISA). Using ELISA, we were able to detect 0.01 g of CSS protein/ml in pure culture. A strong color reaction was observed with CSS antiserum and CSS antigens, whereas withSpiroplasma citri and honeybee spiroplasma (AS-576) antigens the color reaction was very weak. No color reaction was observed with four other spiroplasmas,Mycoplasma gallisepticum, andAcholeplasma laidlawii. Antiserum against CSS with ELISA successfully detected CSS in diseased plants and insect vectors. Host plant and vector tissue had no detrimental effect on the reaction. With ELISA,Spiroplasma citri antiserum did not react positively with CSS-infected plant or insect tissue, whereas a positive color reaction was observed withS. citri-infected (stubborn disease) citrus plant samples.     

Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells

We present a fully defined culture system (adapted Essential8^TM [E8^TM] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8^TM medium was modified by adding (1) l ‐proline, (2) l ‐ornithine, (3) N^ω‐hydroxy‐nor‐l ‐arginine acetate, or by (4) lowering the arginine concentration. Reduction of arginine conversion was best obtained by adding 5 mM l ‐ornithine, followed by 3.5 mM l ‐proline and by lowering the arginine concentration in the medium to 99.5 μM. No major changes in pluripotency and cell amount could be observed for the adapted E8^TM media with ornithine and proline. However, our subsequent ion mobility assisted data‐independent acquisition (high‐definition MS) proteome analysis cautions for ongoing changes in the proteome when aiming at longer term suppression of arginine conversion.     

Isolation and Culture of Corn Stunt Spiroplasma in Serum-Free Medium

A serum-free medium (medium A₆), supports the primary isolation and cultivation in vitro of corn stunt spiroplasma (CSS). The growth characteristics of CSS in A₆ medium are similar to those in the conventional serum dependent, ketoglutarate sorbitol medium (SMK).     

Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity

Isolated single smoothmuscle cells (SMCs) from different regions of the rabbit stomach wereused to determine a possible correlation between unloaded shorteningvelocity and smooth muscle (SM) myosin heavy chain (MHC) S1 headisoform composition (SMA, no head insert; SMB, with head insert).-Toxin-permeabilized isolated single cells were maximally activatedto measure unloaded shortening velocity and subsequently used in anRT-PCR reaction to determine the SMA/SMB content of the same cell. SMMHC SMA and SMB isoforms are uniquely distributed in the stomach with cells from the fundic region expressing little SMB (38.1 ± 7.3% SMB; n = 16); cells from the antrum express primarilySMB (94.9 ± 1.0% SMB; n = 16). Mean fundic cellunloaded shortening velocity was 0.014 ± 0.002 cell lengths/scompared with 0.036 ± 0.002 for the antrum cells. Unloadedshortening velocity in these cells was significantly correlated withtheir percent SMB expression (r² = 0.58).Resting cell length does not correlate with the percent SMB expression(n = 32 cells). Previously published assays of purifiedor expressed SMA and SMB heavy meromyosin show a twofold difference inactin filament sliding speed in in vitro motility assays. Extrapolationof our data to 0-100% SMB would give a 10-fold range ofshortening velocity, which is closer to the ~20-fold range reportedfrom various SM tissues. This suggests that mechanisms in addition tothe MHC S1 head isoforms regulate shortening velocity.

     

Biosynthesis of lipids by Rhodosporidium toruloides ATCC 10788

The limiting amount of nitrogen required to trigger lipid accumulation in the oleaginous yeast Rhodosporidium toruloides ATCC 10788 was studied, batchwise, by subjecting washed mid-exponentially grown cells to nitrogen at levels of 10⁻² M down to 10⁻⁴ M per g l⁻¹ of lean cells (2–5% fat content) in a mineral medium where glucose was present at 35 g l⁻¹. The results showed that lipid accumulation always started sometime after nitrogen reached a level of 3 × 10⁻⁵ M and the specific initial lipid productivity was constant. Furthermore, the cells were subjected to nine combinations of temperature and pH, from (25° C, pH 4.5) to (35° C, pH 7.5) in the mineral medium supplemented with 0.5 g l⁻¹ of yeast extract and 1 g l⁻¹ (NH₄)₂SO₄. As was expected, lipid content in the cells was higher at 25° C, but pH around 6.0–7.5 slightly enhanced the effect of lower temperature. The effect of pH was also noticed to affect the size of changes in the temporal profiles of the oil's fatty acid distribution prior to nitrogen depletion, whereas no significant difference in the fatty acid composition of the oil was shown after exhaustion of nitrogen from the medium for all combinations of temperature and pH.     

Transport of l-arginine in brush border vesicles derived from rabbit kidney cortex

The uptake of l-arginine by brush border vesicles from rabbit kidney cortex was investigated at 37 °C and pH 7.5. The initial rate of uptake (15 s) was twice as fast in a highly purified brush border as in brush border contaminated by basal-lateral plasma membranes. The initial uptake in a mannitol medium can be best described as the sum of transfer by two systems with K_m values of 0.07 and 3.5 mm and V_max values of 1.5 and 8 nmol/mg protein × 15 s, respectively. For the inhibitors of l-[¹⁴C]arginine, uptake (15 s at two substrate concentrations of 0.1 and 2.5 mm in a mannitol medium) the following sequence of inhibitory strength was established: l-arginine, l-ornithine, l-cystine, l-lysine, d-arginine, and NaCl. When a vesicular membrane potential was induced transiently by a jump of the pH in the incubation medium from 5.9 to 7.5 or by an outward movement of K⁺ in the presence of gramicidin D, an overshoot of l-arginine uptake was observed. Initial uptake of l-arginine was slightly faster in the presence of a Na⁺ gradient (outside to inside) than under a K⁺ gradient. Both ion gradients reduced uptake as compared to the uptake in a mannitol medium. Uptake was also studied after the membrane potential was minimized by equilibrating the vesicles in a NaCl or KC1 medium in the presence of gramicidin D. Under these conditions, l-arginine uptake in the first 30 s was faster in the NaCl than in the KCl medium. These experiments indicate, beside a major ion-independent l-arginine transport, the presence of a transport stimulated by Na⁺ in isolated brush border vesicles.     

Phylogenetic characterization of two novel species of the genus Bifidobacterium: Bifidobacterium saimiriisciurei sp. nov. and Bifidobacterium platyrrhinorum sp. nov.

Three bifidobacterial Gram-stain-positive, non-spore forming and fructose-6-phosphate phosphoketolase-positive strains, SMA1^T, SMB2 and SMA15^T were isolated from the faeces of two adult males of the squirrel monkey (Saimiri sciureus).On the basis of 16S rRNA gene sequence similarities, the type strain of Bifidobacterium primatium DSM 100687^T (99.3%; similarity) was the closest neighbour to strains SMA1^T and SMB2, whereas the type strain of Bifidobacterium stellenboschense DSM 23968^T (96.5%) was the closest neighbour to strain SMA15^T. The average nucleotide identity (ANI) values of SMA1^T and SAM15^T with the closely related type strains were 93.7% and 88.1%, respectively. The in silico DNA‒DNA hybridization values with the closest neighbours were 53.1% and 36.9%, respectively. GC contents of strains SMA1^T and SMA15^T were 63.6 and 66.4 mol%, respectively. Based on the phylogenetic, genotypic and phenotypic data obtained, the strains SMA1^T and SMA15^T clearly represent two novel taxa within the genus Bifidobacterium for which the names Bifidobacterium saimiriisciurei sp. nov. (type strain SMA1^T = BCRC 81223^T = NBRC 114049^T = DSM 106020^T) and Bifidobacterium platyrrhinorum sp. nov. (type strain SMA15^T = BCRC 81224^T = NBRC 114051^T = DSM 106029^T) are proposed.     

Acquisition and transmission of corn stunt spiroplasma by its leafhopper vector Dalbulus maidis

As the acquisition access period of Dalbulus maidis on infected maize increased from 15 min to 7 days, the incubation period of corn stunt spiroplasma (CSS) in the insect decreased from 27 days to 8 days and the final proportion of transmitting insects increased from 5% to 100%. After 7 days access the median incubation period (IPsO) was 14.3 days (IP₅₀ females = 12.9 days: IP₅₀ males =16.8 days), while the proportion of transmitting insects increased from 4. 3% (9 days after the start of acquisition access) to a maximum of 93% (after 22 days), before decreasing. Females started transmitting significantly earlier and a greater proportion transmitted each day than males, until day 22 when both sexes transmitted equally. Of the insects which transmitted CSS, 29% did so continuously until death; 66% failed to transmit during the last 1–3 days, and 5% transmitted intermittently towards the end of their life. During daily transfer, females were more likely to infect plants consecutively (up to 25) than males, and females infected the higher proportion of test plants. As the transmission access period was increased from 1 h to 72 h, the proportion of transmitting insects increased from 22.5% to 97.3% and the incubation period in maize decreased.     

Growth kinetics of biopigment production by Thai isolated <Emphasis Type="Italic">Monascus purpureus</Emphasis> in a stirred tank bioreactor

Monascus purpureus is a biopigment-producing fungi whose pigments can be used in many biotechnological and food industries. The growth kinetics of biopigment production were investigated in a liquid fermentation medium in a 5-l stirred tank bioreactor at 30°C, pH 7, for 8 days with 100 rpm agitation and 1.38 × 10⁵ N/m² aeration. Thai Monascus purpureus strains TISTR 3002, 3180, 3090 and 3385 were studied for color production, growth kinetics and productivity. Citrinin as a toxic metabolite was measured from the Monascus fermentation broth. The biopigment productions were detected from fermentation broth by scanning spectra of each strain produced. Results showed a mixture of yellow, orange and red pigments with absorption peaks of pigments occurring at different wavelengths for the four strains. It was found that for each pigment color, the color production from the strains increased in the order TISTR 3002, 3180, 3090, 3385 with 3385 production being approximately 10 times that of 3002. Similar results were found for growth kinetics and productivity. HPLC results showed that citrinin was not produced under the culture conditions of this study. The L*, a* and b* values of the CIELAB color system were also obtained for the yellow, orange and red pigments produced from the TISTR 3002, 3180, 3090 and 3385 strains. The colors of the pigments ranged from burnt umber to deep red.     

Development of isolation and cultivation method for outer root sheath cells from human hair follicle and construction of bioartificial skin

Obtaining a sufficient amount of healthy keratinocytes from a small tissue is difficult. However, ORS cells can be a good source of epithelium since they are easily obtainable and patients do not have to suffer from scar formation at donor sites. Accordingly, the current study modified the conventional primary culture technique to overcome the low propagation and easy aging of epithelial cells during culturing. In a conventional primary culture, the average yield of human ORS cells is 2.1×10³ cells/follicle based on direct incubation in a trypsin (0.1%)/EDTA (0.02%) solution for 15 min at 37°C, however, our modified method was able to obtain about 6.9×10³ cells/follicle using a two-step enzyme digestion method involving dispase (1.2 U/mL) and a trypsin (0.1%)/EDTA (0.02%) solution. Thus, the yield of primary cultured ORS cells could be increasd three times higher. Furthermore, a total of 2.0×10⁷ cells was obtained in a serum-free medium, while a modified E-medium with mitomycin C-treated feeder cells produced a total of 6.3×10⁷ cells over 17 days when starting with 7.5×10⁴ cells. Finally, we confirmed the effectiveness of our ORS cell isolation method by presenting their ability for reconstructing the bioartificial skin epitheliumin vitro     

Direct isolation in cell-free medium of a spiroplasma fromHaemaphysalis leporispalustris (Acari: Ixodidae) in Maryland

A spiroplasma isolate, was obtained from rabbit ticks (Haemaphysalis leporispalustris) taken from cottontail rabbits in Maryland by inoculation of tick suspensions into SP-4 medium. The isolate was indistinguishable from an experimental vertebrate pathogen (suckling mouse cataract agent spiroplasma) when tested with other plant and tick spiroplasmas in growth inhibition, deformation, and metabolism inhibition tests. The isolated organism had a pathogenic profile for suckling rats and embryonated chicken eggs that differed significantly from that of other suckling mouse cataract agent strains. This is the first report of a direct spiroplasma isolation from ticks in cell-free medium, and confirms the specific association of spiroplasmas of the suckling mouse cataract agent serogroup with rabbit ticks.     

制备可溶性肿瘤坏死因子受体II－脂联素球部融合蛋白的两种细胞培养工艺比较

利用7.5 L生物反应器篮式贴壁培养和全悬浮批式培养CHO工程细胞株表达可溶性肿瘤坏死因子受体Ⅱ-脂联素球部(sTNFRⅡ-gAD)融合蛋白,比较这两种培养方法的产率,以便优化高效表达sTNFRⅡ-gAD融合蛋白的制备工艺.篮式贴壁培养首先小规模培养CHO工程细胞株,待细胞增殖到一定密度后以3× 105～4× 105 cells/mL密度接种生物反应器贴壁培养3d,调换成不含血清的LK021培养基继续培养4d.而全悬浮无血清批式培养则以3×105～4×105 cells/mL密度的CHO工程细胞株接种于生物反应器,连续培养7d.培养过程实时监测培养条件,维持pH和DO的稳定.分别收集细胞上清,离心去细胞后用Pellicon切相流超滤系统对蛋白进行浓缩,并通过DEAE离子交换柱进行纯化.结果显示,篮式贴壁培养和全悬浮批式培养均成功表达了sTNFRⅡ-gAD融合蛋白,产量分别为8.0 mg/L和7.5 mg/L、纯度分别为95％和98％,从而为sTNFRⅡ-gAD融合蛋白的中试工艺研究提供了一定的基础.     

Influence of humidity on the infection of western flower thrips, <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> (Thysanoptera: Thripidae), by <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

The insecticidal activity of Beauveria bassiana GHA derived from a commercial mycoinsecticide BotaniGard ES against Frankliniella occidentalis was determined in a bioassay by dipping the female adults into a conidial suspension. The 90% lethal concentration of B. bassiana GHA was estimated to be 9.7 × 10⁶ conidia/ml. The lethal times for achieving 90% mortality of thrips inoculated with a 1/500-diluted solution of BotaniGard ES and a 10^7.5 (3.16 × 10⁷) conidia/ml suspension of B. bassiana GHA were estimated to be five and six days, respectively. When the treated thrips were exposed to a high relative humidity (RH) of over 99% for various periods and then transferred to 60% RH, the requisite lengths of the high-humidity period to achieve 90% mortality of the thrips at six days after inoculation were estimated to be 46 and 47 h in BotaniGard ES and B. bassiana GHA, respectively. Fungal multiplication in the thrips was detected between 48 to 60 h after inoculation by measuring Beauveria-specific DNA in the host following inoculation with a B. bassiana GHA suspension of 10^7.5 conidia/ml using a real-time quantitative PCR. The mycelial growth in the host hemocoel was not influenced by the low-humidity condition.     

TubeSpin bioreactor 50 for the high-density cultivation of Sf-9 insect cells in suspension

Here we present the TubeSpin bioreactor 50 (TubeSpins) as a simple and disposable culture system for Sf-9 insect cells in suspension. Sf-9 cells had substantially better growth in TubeSpins than in spinner flasks. After inoculation with 10⁶ cells/ml, maximal cell densities of 16 × 10⁶ and 6 × 10⁶ cells/ml were reached in TubeSpins and spinner flasks, respectively. In addition the cell viability in these batch cultures remained above 90% for 10 days in TubeSpins but only for 4 days in spinner flasks. Inoculation at even higher cell densities reduced the duration of the lag phase. After inoculation at 2.5 × 10⁶ cells/ml, the culture reached the maximum cell density within 3 days instead of 7 days as observed for inoculation with 10⁶ cells/ml. Infection of Sf-9 cells in TubeSpins or spinner flasks with a recombinant baculovirus coding for green fluorescent protein (GFP) resulted in similar GFP-specific fluorescence levels. TubeSpins are thus an attractive option for the small-scale cultivation of Sf-9 cells in suspension and for baculovirus-mediated recombinant protein production.     

Carbon Dioxide and pH Requirements of Non-Photosynthetic Tissue Culture Cells

Maximum growth of suspension cultures of Paul's Scarlet rose required a low pH (5.2 to 5.4) during the division phase (day 0 to 7) and a higher pH (5.8 to 6.0) during the expansion phase (day 7 to 14). The fresh weight increase was reduced by approximately 22%, but the dry weight was not influenced when cells were grown for 14 days in a CO₂ deficient environment. Kinetic studies showed that the first five days of growth was the critical period of nonautotrophic CO₂ fixation when cells were grown in medium buffered at pH 5.4. The phosphoenolpyruvate carboxylase activity was highest (0.50 × 10⁶ cpm min^?1· g^?1 fresh weight) during the period when nonautotrophic CO₂ fixation appeared to be critical for growth.     

Factors influencing the growth and respiration of rat cerebral astrocytes in primary culture

Cell densities and respiratory rates of astrocytes from neonatal rat brain grown in primary culture were determined after 20–30 days in vitro. Cells grown in flasks reached lower densities (g DNA/cm²) and higher protein: DNA ratios than cells grown in petri dishes. Respiratory rates were lower for cells grown in flasks compared to cells grown in dishes. The pH of the medium in flasks fell below 6.9 between feedings while the pH of the medium in dishes remained at about 7.2. Cells grown in dishes with the medium pH adjusted to 6.8 also showed lower final cell densities, higher protein: DNA ratios, and lower respiratory rates, compared to cells grown under similar conditions at pH 7.5. Intermediate values of each parameter were found in cells grown at pH 7.5 for one week and then at 6.8 for 20 days. We conclude that the effects of ambient pH account for the differences in growth characteristics and respiratory rates of astrocytes grown in dishes versus those grown in flasks.     

Improvement of liver fibrosis by infusion of cultured cells derived from human bone marrow

We develop “autologous bone marrow cell infusion (ABMi) therapy” for the treatment of human decompensated liver cirrhosis and confirm the efficacy and safety of this treatment in multicenter clinical studies. With the goal of further expanding the applications of ABMi, we first cultured human bone marrow cells and then determined whether a cell fraction found to be effective in improving liver fibrosis can be amplified. Cells harvested after two passages (P2 cells) consistently contained approximately 94 % mesenchymal stem cells (MSCs); conversely, the cells harvested after only medium change (P0 cells) contained many macrophages. MSCs (2.8?×?10⁸) in P2 cells were harvested from 3.8?×?10⁸ bone marrow-derived mononuclear cells after 22 days. DNA-chip analysis also showed during the culturing step that bone marrow-derived cells decreased with macrophage phenotype. The infused 5?×?10⁵ P2 cells significantly improved liver fibrosis in the nonobese diabetic/severe combined immunodeficient (NOD-SCID) mouse carbon tetrachloride (CCl₄) liver cirrhosis model and induced the expression of matrix metalloproteinase (MMP)-9 and suppressed expressions of alpha smooth muscle actin (αSMA), tumor necrosis factor alpha (TNFα) and transforming growth factor beta (TGFβ) in the liver. Cultured human bone marrow-derived cells (P2 cells) significantly inhibited liver fibrosis. The increase of MMP-9 and suppressed activation of hepatic stellate cells (HSCs) through the regulation of humoral factors (TNFα and TGFβ) contribute to the improvement of liver fibrosis by MSCs comprising about 94 % of P2 cells. MSCs in cultured human bone marrow-derived mono-nuclear cells (BM-MNCs) proliferate sufficiently in cell therapy, so we believe our cultured bone marrow-derived cell therapy can lead to expanded clinical applications and enable outpatient therapy.