A novel biosynthesis of medium chain length alpha-ketodicarboxylic acids in methanogenic archaebacteria

Gas chromatographic-mass spectrometric analysis on the distribution of alpha-ketodicarboxylic acids in various bacteria determined that alpha-ketoglutarate and alpha-ketoadipate are widely distributed in all the bacteria examined, whereas alpha-ketopimelate and alpha-ketosuberate are found only in the methanogenic archaebacteria. Labeling experiments with stable isotopes indicated that each of these acids arises from alpha-ketoglutarate by repeated alpha-ketoacid chain elongation. The final product in this series of reactions, alpha-ketosuberate, serves in the methanogenic bacteria as the biosynthetic precursor to the 7-mercaptoheptanoic acid portion of 7-mercaptoheptanoylthreonine phosphate, a methanogenic coenzyme.     

Improvement of a synthetic medium for Dictyostelium discoideum

Dictyostelium discoideum is of considerable interest as an expression system for the production of proteins of high value. The cultivation of this social amoeba is not as easy as that of other common microbial expression systems. Wildtype strains grow on bacteria. Mutant strains growing on axenic media reach cell densities of 1–2×10⁷ ml⁻¹ when cultivated in commonly used complex media. A totally synthetic medium formulated by Franke and Kessin (Proc. Natl. Acad. Sci. USA 74 (1977) 2157) has become popular and allows cell densities of about 3×10⁷ ml⁻¹ to be obtained. This medium (FM) is being improved mainly on the basis of the analysis of limitations with respect to amino acids. With this improved synthetic medium (SIH) cell densities in the order of 5–6×10⁷ ml⁻¹ have been achieved.     

GABA interaction with lipids in organic medium

The interaction of 3H-GABA (gamma-aminobutyric acid and 14C-glutamate with lipids in an aqueous organic partition system was studied. With this partition system 3H-GABA and 14C-glutamate were able to interact with sphingomyelin, sulfatide, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid but not with cholesterol or ceramide. In an homogeneous aqueous medium we could not demonstrate any interaction between 3H-GABA and lipids. The apparent dissociation constants (Kd) for 3H-GABA-lipids or 14C-glutamate-lipids interactions in organic medium were in the millimolar range and maximal charge (Bmax) between 3 and 7 moles of GABA or glutamate by mole of lipid. Amino acids such as glutamic acid, beta-alanine and glycine displaced 3H-GABA with the same potency as GABA itself; thus these results show that the interaction lacks pharmacological specificity. To detect this interaction lipid concentrations higher than 2 microM were required and in the partition system 3H-GABA and lipid phosphorus were both concentrated at the interface. Therefore lipids tested with a biphasic partition system do not fulfill the classical criteria for a neurotransmitter receptor at least not for GABA and glutamate.     

Behaviour ofPropionibacterium shermanii in alkaline medium

Physiological and morphological properties ofPropionibacterium shermanii were estimated in a liquid semisynthetic medium of initial pH between 7 – 10. Prolongation of the lag phase and a shift of the stationary phase occurred at higher initial pH values of the medium, The growth response of the strain depended on the way of glucose addition (either sterilized in the medium or added aseptically after sterilization). At pH 8.5 and higher the cells begin to form slime and capsules. The strain exhibited growth activity even at initial pH values of the medium 11.5.     

Morphological and biochemical differentiation of limb bud cells cultured in chemically defined medium

When chick limb buds were isolated from stage 22–23 embryos and cultured in chemically defined medium “BGJb,” the explants grew and, after about 9 days, showed good chondrogenesis of recognizable cartilage segments. Cartilage-type proteoglycan (termed PCS-H) was not synthesized during early days of culture, but by Day 9, it became a major proteoglycan constituent of the tissue. Freshly dissociated limb bud cells, when plated as monodispersed cultures at a density of 7 × 10⁶ cells/ml of BGJb, did not undergo chondrogenic differentiation and, instead, assumed the appearance of unhealthy or degenerated cells. During 9 days of culture, even though proteoglycans were synthesized, they were nevertheless of much smaller molecular size than PCS-H. When limb bud cells were cultured as a pellet containing 7 × 10⁶ cells in 1 ml of BGJb, a more tightly packed aggregate of about 2 × 10⁶ cells appeared in an inner region of the pullet during the first 24 hr of culture, and by Day 12 the aggregate had differentiated into a cartilage nodule surrounded by a thin layer of what appear to be ectodermal cells. As the conversion of aggregate into cartilage nodule progressed, newly formed proteoglycans gradually became more like cartilage-type proteoglycans, and by Day 12 they had many chemical and physical characteristics similar to those of the proteoglycans isolated from fully differentiated cartilages. The results indicate that the initial association of limb bud cells is an important factor for the chondrogenesis in BGJb and further suggest that the tight binding of the cell surfaces to one another may directly or indirectly stimulate the mechanism of synthesis of cartilage-type proteoglycans.     

Microwave-assisted preparation of affinity medium

Microwave assistance was used for preparing polyethylene glycol (PEG)-Cibacron blue 3GA and Sepharose CL-4B-Cibacron blue 3GA affinity materials. The former was used as the affinity macroligand in a PEG-dextran aqueous two-phase system for purification of alcohol dehydrogenase and EcoRI. The Sepharose CL-4B-Cibacron blue 3GA was used for affinity chromatography of the above two enzymes. It was found that microwave assistance could reduce the time of PEG-dye preparation to 5 min (from 7h). Similarly, Sepharose CL-4B-Cibacron blue 3GA preparation time could be reduced to 21 min (from 3.5h). The performances of affinity macroligand PEG-dye and the affinity medium Sepharose-dye prepared by conventional methods and with microwave assistance were similar during purification of these enzymes.     

漏斗多孔菌液体菌种培养基及其栽培条件

为实现漏斗多孔菌资源化利用,设计单因素试验,以菌丝生物量、菌球密度和菌球直径为指标,获得漏斗多孔菌Polyporus arcularius液体菌种培养基配方为马铃薯（去皮）200g、玉米粉20.0g、蛋白胨5.0g、KH₂PO₄ 3.0g、K₂HPO₄ 1.0g,MgSO₄·7H₂O 1.5g,初始pH 5.0,并优化培养条件为装液量250mL/500mL,接种量2%,培养温度32℃,转速170r/min,种龄6d;依上述条件制备液体菌种,以固体菌种为对照,进行出菇试验,调查农艺性状,试验结果表明液体菌种接种后,菌袋满袋时间提前7d,子实体原基产生时间提前8d,头潮菇鲜重提高22.5%;配方试验结果表明,以细木屑为主料的配方产量最高,头潮菇鲜重为270.6g/袋。     

双歧杆菌番茄培养基的初步研究

目的 研制双歧杆菌番茄培养基,为后期双歧杆菌发酵剂的研究奠定基础.方法 以番茄汁为基础,采用单次单因子法与Plackett-Burman( P-B)设计法相结合,对BL培养基进行优化.结果 将BL培养基的16种成分筛选为7种,即葡萄糖、多胨、胰胨、异麦芽寡糖、可溶性淀粉、磷酸二氢钾和番茄汁,为获得产菌量高、成本 低、制作工艺简单的双歧杆菌番茄培养基奠定了基础.结论 将传统的非统计法和统计法相结合,既可以降低生产成本,又能在制作过程中节约劳动力.     

Production of rosamicin: improvement of synthetic medium

Rosamicin is one of the important macrolide antibiotics that has clinical efficacy and broad-spectrum antibacterial activity. Using a mutant strain of Micromonospora rosaria (NRRL 3718), a chemically defined medium was developed, and some fermentation conditions that are important to rosamicin biosynthesis were optimized to achieve rosamicin productivity of 230 mug/ml. Soluble starch and l-asparagine were found to be the best carbon and nitrogen sources, and a stimulative effect of magnesium and zinc ions was also found. The medium developed contains: soluble starch, 4%; l-asparagine, 0.15%; K(2)HPO(4), 0.075%; CaCO(3), 0.6%; MgSO(4) . 7H(2)O, 0.05%; FeSO(4) . 7H(2)O, 10 M; CuSO(4) . 5H(2)O, 10 M; ZnSO(4) . 7H(2)O, 10 M; and MnSO(4) . (4-6)H(2)O, 10 M. The required air supply was about 40 mmol of O(2) liter . h . atm, and the favorable culture temperature was 28 to 29 degrees C.     

Variation in resistance of Mycobacterium paratuberculosis to acid environments as a function of culture medium

Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20 degrees C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log(10) concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.     

Optimization of Bacillus subtilis natto growth parameters in glycerol-based medium for vitamin K (Menaquinone-7) production in biofilm reactors

Bioprocess and Biosystems Engineering - Menaquinone-7 (MK-7) is the key form of vitamin K used as a dietary supplement and its production revolves around Bacillus subtilis natto. Current...     

Evaluation of m-T7 agar as a fecal coliform medium

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Decreased ouabain binding in cystic fibrosis fibroblasts in potassium-free medium

We have previously demonstrated that cystic fibrosis (CF) cells show increased survival compared to normal cells when exposed to ouabain in medium lacking potassium. In this report, we show that the CF cells bind significantly less ouabain than the normal cells in potassium-deficient medium. Using age-matched normal and CF skin fibroblast strains, we show that (1) at ouabain concentrations <20 × 10⁻⁹ M binding for both normal and CF cells is linear with time for 1 h and reaches equilibrium after 4 h; (2) at ouabain concentrations between 2 and 20 × 10^-9 M, the initial rate of binding for CF cells is approx. 70% of the normal cells; (3) under equilibrium-binding conditions, Scatchard analysis reveals that three different CF strains have 12, 16, and 44% fewer ouabain-binding sites than their matched normal controls. In addition, studies in potassium-free medium of the inhibition of ⁸⁶Rb flux (a K⁺ analogue) into the cell by ouabain show no differences between CF and normal cells. We have also previously shown that in a low glucose, potassium-deficient medium the CF cells survived ouabain exposure no better than normal cells. In this report, equilibrium-binding studies with [³H]ouabain clearly show that CF cells bind less ouabain under these conditions than normal cells. These results indicate that ouabain resistance in CF cells is not solely a function of differences in ouabain binding. Furthermore, the differential ouabain killing may not be due to ion transport differences, but rather to as yet unknown mechanisms. CF cells thus appear to be unlike previously characterized ouabain-resistant mutants.     

Effect of preameloblast-conditioned medium and CPNE7 on root surfaces in dogs: a histologic and histomorphometric evaluation

Preameloblast-conditioned medium (PACM) has been reported as a potent dentin regenerative material, but its effects as a mixture on periodontal regeneration and the role of CPNE7 in PACM are not known. The purpose of this study is to evaluate the histologic and histomorphometric effects of preameloblast-conditioned medium (PACM) and CPNE7 on periodontal tissue healing in dogs. Seventy-two mandibular premolar roots from ten dogs were extracted and randomly divided into six groups (n?=?12 each): (1) positive control group; (2) negative control group; (3) cementum-removed and PACM-treated group; (4) cementum-preserved and PACM-treated group; (5) CPNE7-inactivated PACM-treated group; and (6) recombinant CPNE7-treated group. The extracted roots were replanted into extraction sockets for 4 and 8 weeks and analyzed histologically. Most of the root surfaces in the negative control group showed ankylosis; and those in the experimental groups showed newly formed PDL-like and cementum-like tissues. Histomorphometric analysis of horizontal sections showed that the mean length of the PDL on the roots of the positive controls was similar to those in cementum-removed or -preserved and PACM-treated group at 8 weeks (p?=?1.08). Sagittal sections showed that the mean length of the new cementum on the roots in cementum-removed and PACM-treated group was significantly greater than that in CPNE7-inactivated PACM-treated group (p?=?0.037). The mean length of the newly formed PDL on the roots in CPNE7- inactivated PACM-treated and rCPNE7-treated groups was significantly greater than that in the negative controls at 8 weeks (p?=?0.037, p?=?0.036). The use of PACM and CPNE7 in tooth replantation resulted in increased PDL and cementum formation, suggesting the beneficial role of PACM and CPNE7 in periodontal tissue healing.     

Evaluation of m-T7 agar as a fecal coliform medium.

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Proliferation of 3T3-L1 preadipocytes in a completely defined serum-free medium

We have developed a completely defined serum-free medium that supports the growth of Swiss 3T3-L1 fibroblasts to nearly the same extent as Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. With ASF301 medium [former name, RITC 80-7; Yamane et al. Exp. Cell Res. 134, 470 (1981)], most of the 3T3-L1 cells survived for at least 10 days, but did not grow. ASF301 medium contains insulin and mouse-epidermal growth factor as growth factors, which are termed "progression factors". So we examined the effects of "competent factors" on the proliferation of 3T3-L1 preadipocytes. Growth in the medium supplemented with competent factors reached a confluent monolayer in 6-7 days. Furthermore, it was confirmed that 3T3-L1 cells grown in the serum-free medium retained the properties of differentiation into adipocytes. Our serum-free medium should be a useful tool for research on the growth and differentiation of 3T3-L1 preadipocytes.     

Radiation quality of tritium beta-rays: microdosimetric distributions for nanometer-size targets in a medium containing tritiated water

To elucidate the characteristics of the action of tritium beta-rays, the following parameters are derived: electron slowing down spectra of primary electrons (beta-rays) and delta-rays in a medium containing tritiated water; and frequency distributions for the microdosimetric quantity j (number of effective primary events per track per target), fj, for nanometer-size targets exposed to tritiated water. Features of the radiation quality of tritium beta-rays are discussed by comparing the present results with those for 60Co gamma-rays and 7 MeV electrons. It is concluded that, although tritium beta-rays, 60Co gamma-rays, and 7 MeV electrons are classified as the same low l.e.t. radiation, the radiation quality of tritium beta-rays is considerably different from those of 60Co gamma-rays and 7 MeV electrons, and has specific features such as a high average l.e.t., a small total electron fluence per unit absorbed dose, and a different microdosimetric distribution, fj, for nanometer-size targets.     

Production of functional hepatocyte growth factor (HGF) in insect cells infected with an HGF-recombinant baculovirus in a serum-free medium

Three insect cell lines, SL-7B cells derived from Spodoptera litura, Sf9, and High Five (Hi-5) cells, were used for the production of pro-hepatocyte growth factor (pro-HGF). Cells were cultured and then infected with a recombinant HGF-containing baculovirus in a serum-free medium. In SL-7B cells, pro-HGF is synthesized and excreted from the cells and late in infection is converted to a heterodimeric form of HGF even when the cells are grown in serum free medium. Conversion of a single-chain form of HGF (pro-HGF) into an HGF heterodimer was unexpected, as pro-HGF is normally cleaved by a serum protease called HGF activator. The proliferation activity of heparin-affinity-purified HGF from serum-free culture supernatant of SL-7B cells is comparable to that obtained from HGF converted by serum proteases, suggesting that SL-7B cells produce a functionally analogous protease to correctly process pro-HGF. This work reports, for the first time, on the feasibility of properly processing pro-HGF to form functional HGF by proteases from invertebrate cells in serum-free media. Avoiding the supplementation of sera provides the advantages of a low production cost, zero contamination of infectious agents from sera, and simple downstream product purification. Experimental results further demonstrate that the conversion of pro-HGF by insect cells is cell-line-dependent, because proteases in Hi-5 or Sf9 cells could not process pro-HGF as efficiently and properly as those in SL-7B cells.     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。     

Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca²⁺-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for α-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as α-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.