Prostacyclin synthesis by endothelial cells from human umbilical veins: effect of cumulative population doublings

There is a wide range of reported values for prostacyclin (PGI2) synthesis by cultured endothelial cells from human umbilical veins (HUVE). Part of this variation may be due to differences in isolation and culture conditions, but part may be due to previously unstudied variation in the number of population doublings (PDs) which the cells have undergone in vitro. Attention is now shifting to arachidonic acid (AA) metabolism by cells from adult human vessels and these cells may require increased PDs to obtain confluent cultures for testing. Therefore, we have examined the effect of number of cell population doublings as well as number of subcultivations on PGI2 synthesis using HUVE as a model system. Primary and first subcultivation cultures inoculated at high density, so that PDs at confluence were less than 4, synthesized 10 times as much PGI2 as the same isolates inoculated at low density with PDs greater than 4. Isolates inoculated and subcultivated so that the PDs at confluence after the fourth subcultivation were less than 6, showed 50% less PGI2 synthesis between the primary and first subcultivation and between the first and second subcultivations. Isolates with less than 4 PDs after the fourth subcultivation were carried further to determine the effect of extensive subcultivation. Four of six isolates showed a sudden increase in PGI2 synthesis which occurred between subcultivations 5 and 12 (PDs 4-6). These results demonstrate that AA metabolism is markedly affected by growth in culture and serial subcultivation.     

Factors influencing microbiological assay of cell-culture mycoplasma.

A total of 6432 cell cultures was assayed for mycoplasmas over a 6-year period by aerobic and anaerobic incubation of agar and broth media. Mycoplasmas were detected in 375 cultures (5.8%). M. orale and A. laidlawii accounted for 61.3% of the isolates. Anaerobic incubation detected 98.1% of the isolates; aeorbic incubation detected 45.8%. Of factors studied to determine their effect on mycoplasma assay, only two, anaerobic incubation and presence of mycoplasmacidal/static antibiotics, were significant. In separate studies, 86 of 2656 cell cultures (3.2%) were infected with strains of M. hyorhinis that did not grow on cell-free media. Recommendations are given for microbiological assay of cell-culture mycoplasmas.     

A Serologic Variant of Mycoplasma Hyorhinis Recovered from the Conjunctiva of Swine

In an examination of conjunctival samples from 40 piglets for mycoplasmas, 17 isolates were obtained. Eight could be identified as Mycoplasma hyorhinis, three as Mycoplasma flocculare, and one as Acholenlasma sp. Five strains were not readily identifiable, but together with two previously recovered strains they were found to represent a distinct serogroup. All seven strains were glucose and phosphatase positive. Incubation in a CO2-enriched atmosphere led to enhancement of the growth on solid medium. The serogroup was serologically related to M. hyorhinis, but not to a number of other glucose fermenting species of mycoplasma, and it may therefore be regarded as a new subspecies of M. hyorhinis.     

Mycoplasmal infection of insect cell cultures

Twenty-five cell cultures of three insect orders from eight laboratories were tested for mycoplasmal infection. Acholeplasma laidlawii was detected in one culture, an incidence of 4.0%. A. laidlawii, Mycoplasma orale, M. arginini, but not M. hyorhinis, could establish infections of drosophila Dm-1 cell cultures at 25 degrees C. In prospective studies, drosophila Dm-1 cultures were intentionally infected with broth-propagated A. laidlawii and M. hyorhinis. M. hyorhinis did not grow and was eliminated from the Dm-1 cultures during consecutive passages. A. laidlawii grew without obvious cytopathic effects during six weekly passages; titers of over 10(7) CFU/ml were recorded at Passages 2 and 5 (p2 and p5). Minimal cell culture infectious doses were also determined during these studies. 0.1 milliliter cell samples were inoculated into Leighton tubes containing either fresh M1A culture medium or 3T6 indicator cells in McCoy's 5a medium. After 4 d of incubation at 25 and 37 degrees C, respectively, the cover slips were stained by DNA fluorochrome Hoechst 33258 (A. laidlawii) or by specific fluorescein-conjugated antiserum (M. hyorhinis). At p2 with both mycoplasma species, the procedure using M1A medium and incubation at 25 degrees C without 3T6 cells was inferior to indicator cells. In five of six experiments at least a two-log higher titer of mycoplasmas was needed to be detected with M1A and 25 degrees C. At p5 no difference could be found. Uridine phosphorylase assays of Dm-1 cultures infected with A. laidlawii, M. hyorhinis, M. orale, and M. arginini gave clearly positive results only with A. laidlawii. The ratio of incorporated uridine to incorporated uracil method yielded false positives with two drosophila cell lines. Suggestions for assay of mycoplasmas in invertebrate cell cultures are given.     

Occurrence of carotenoids and sporopollenin in Nanochlorum eucaryotum, a novel marine alga with unusual characteristics

Pigment analysis of Nanochlorum eucaryotum on two strains grown under different gaseous conditions was performed. Air-gassed control cultures did not differ qualitatively with respect to the content of chlorophylls a and b, carotenes alpha and beta, lutein, violaxanthin, neoxanthin and cryptoxanthin in comparison with cultures grown under natural gas. The absolute pigment content per cell increased in cultures grown with natural gas. Growth of N. eucaryotum depends on CO2 which is present in concentrations up to 2.0 vol% in natural gas. N. eucaryotum cannot utilize methane and is therefore not methylotrophic. In cultures of N. eucaryotum grown with natural gas and in air-gassed cultures under nitrogen deficient conditions the secondary carotenoids canthaxanthin and astaxanthin could be detected. In air-gassed cultures of strain N. eucaryotum Colona the same secondary carotenoids have been found, while secondary carotenoids were never found in strain N. eucaryotum Mainz. Cell walls of N. eucaryotum always contain sporopollenin as confirmed by isolation, elemental analysis, infrared absorption spectrophotometry, acetolysis-resistance and electron microscopy.     

Aerobic nitrification-denitrification by heterotrophic Bacillus strains

Twenty-four Bacillus strains predominantly outgrown in a night soil treatment system were isolated and characterized. Under various culture conditions, cell interactions took place among them and cell population changed. Maximum removal of NH4+-N and cell production by the isolates occurred under the conditions of 30% DO and C/N ratio of 8. Five dominant isolates were identified to be species of Bacillus cereus, Bacillus subtilis and Bacillus licheniformis with similarities of 78-94%. Additions of 0.8% peptone and 0.3% yeast extract to a basal medium influenced the growth of isolates and the removal of NH4+-N in flask culture. Metal ions such as Ca2+, Fe2+ and Mg2+ had a similar effect. The specific growth rates of the five isolates were found to be in a range of 0.43-0.55 h(-1). During the flask experiment of nitrogen removal under aerobic growth conditions, active nitrification by the isolates occurred largely in 1h with a decrease of COD and alkalinity reduced to only 74.6% of theoretical value. From the nitrogen balance, the percentage of nitrogen lost in the flask culture was estimated to be 33.0%, which was presumed to convert to N2 gas. This conversion of ammonia to N2 without formation of nitrous oxide under aerobic growth conditions was confirmed by GC analysis. From all the results, it has been found that the Bacillus strains were able to occur simultaneously aerobic nitrification/denitrification and the B3 process using the Bacillus strains seemed to possess some economic advantages.     

Regulation of angiotensin I-converting enzyme activity in serially cultivated bovine endothelial cells

Angiotensin I-converting enzyme (ACE) activity was measured in lysates of cloned and uncloned cultures of bovine fetal aortic endothelial cells. The expression of ACE activity in these cells was complex, and influenced by subcultivation, cell density, serum, cumulative population doublings, and clonal heterogeneity. The ACE specific activity at any point in the in vitro lifespan was determined, at least in part, by interaction of these culture variables. After subcultivation to subconfluent densities, cellular ACE specific activity decreased markedly and did not reach detectable levels until cells attained confluent densities. The use of different suppliers' lots of serum in the growth medium resulted in different cellular ACE specific activities. The ACE specific activity decreased as cultures were serially subcultivated, but remained detectable throughout the lifespan, suggesting a linkage between the proliferative history of an endothelial cell and its remaining capacity to express ACE. Increased ACE activity was observed when cells at the end of their lifespan were cultured at high densities. Cloned strains behaved similarly to the uncloned parent culture, except that they exhibited a wide range of ACE specific activities.     

Rapid tissue culture method for detection of Mycoplasma hyorhinis

An easy, rapid tissue culture method for detection of Mycoplasma hyorhinis is described. The organism induces morphological changes in mink S + L - cells. This effect was not observed in eight other animal cell lines infected by M. hyorhinis and it did not occur in the mink cells infected with six other strains of mycoplasma. This cell system should be useful in research laboratories which do not have other standard techniques available for monitoring the presence of M. hyorhinis.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

Characterization of gentamicin-resistant Mycoplasma hyorhinis.

A study was conducted to determine if a gentamicin-resistant strain of mycoplasma could be developed for use in validating current mycoplasma detection methods for biologic product harvest cell culture fluid (CCF) containing gentamicin. A strain of gentamicin-resistant Mycoplasma hyorhinis was isolated and characterized. The study showed that this organism was similar to the wild-type strain in all ways examined except gentamicin resistance. Both strains of mycoplasma (the gentamicin resistant and the wild-type) exhibited comparable growth patterns and showed 100% homology based on DNA sequencing and analysis of a 464-bp PCR product. Also, analysis using species-specific antisera identified both strains as M. hyorhinis. Two commonly used lot release mycoplasma detection methods (culture and DNAF) consistently detected mycoplasmas in spiked biologic product harvest CCF containing gentamicin but not in unspiked samples. This study demonstrates the first isolation and characterization of a gentamicin-resistant M. hyorhinis that can be used to validate mycoplasma detection methods for biologic product harvest CCF containing gentamicin.     

Radiation resistance of lactobacilli isolated from radurized meat relative to growth and environment.

Of 113 lactobacilli isolated from radurized (5 kGy) minced meat, 7 Lactobacillus sake strains, 1 L. curvatus strain, and 1 L. farciminis strain were used for radiation resistance studies in a semisynthetic substrate (i.e., modified MRS broth). Five reference Lactobacillus spp., one Staphylococcus aureus strain, and one Salmonella typhimurium strain were used for comparative purposes. All L. sake isolates exhibited the phenomenon of being more resistant to gamma-irradiation in the exponential (log) phase than in the stationary phase of their growth cycles by a factor of 28%. Four references strains also exhibited this phenomenon, with L. sake (DSM 20017) showing a 68% increase in resistance in the log phase over the stationary phase. This phenomenon was not common to all bacteria tested and is not common to all strains with high radiation resistance. Four L. sake isolates and three reference strains were used in radiation sensitivity testing in a natural food system (i.e., meat). The bacteria were irradiated in minced meat and packaged under four different conditions (air, vacuum, CO2, and N2). Organisms exhibited the highest death rate (lowest D10 values [doses required to reduce the logarithm of the bacterial population by 1] ) under CO2 packaging conditions, but resistance to irradiation was increased under N2. The D10 values of the isolates were generally greater than those of the reference strains. The D10 values were also higher (approximately two times) in meat than in semisynthetic growth medium.     

Radiation resistance of lactobacilli isolated from radurized meat relative to growth and environment

Of 113 lactobacilli isolated from radurized (5 kGy) minced meat, 7 Lactobacillus sake strains, 1 L. curvatus strain, and 1 L. farciminis strain were used for radiation resistance studies in a semisynthetic substrate (i.e., modified MRS broth). Five reference Lactobacillus spp., one Staphylococcus aureus strain, and one Salmonella typhimurium strain were used for comparative purposes. All L. sake isolates exhibited the phenomenon of being more resistant to gamma-irradiation in the exponential (log) phase than in the stationary phase of their growth cycles by a factor of 28%. Four references strains also exhibited this phenomenon, with L. sake (DSM 20017) showing a 68% increase in resistance in the log phase over the stationary phase. This phenomenon was not common to all bacteria tested and is not common to all strains with high radiation resistance. Four L. sake isolates and three reference strains were used in radiation sensitivity testing in a natural food system (i.e., meat). The bacteria were irradiated in minced meat and packaged under four different conditions (air, vacuum, CO2, and N2). Organisms exhibited the highest death rate (lowest D10 values [doses required to reduce the logarithm of the bacterial population by 1] ) under CO2 packaging conditions, but resistance to irradiation was increased under N2. The D10 values of the isolates were generally greater than those of the reference strains. The D10 values were also higher (approximately two times) in meat than in semisynthetic growth medium.     

The Effect of Porcine Mycoplasmas on Pig Tracheal Organ Cultures

S ummary : Tracheal organ cultures were made from 2–3 week old pigs. Tracheal rings were used to assess the pathogenicity of Mycoplasma hyopneumoniae, M. hyorhinis, M. granularum and M. hyosynoviae. Both serum-resistant and serum-sensitive strains of M. hyorhinis had a marked effect on ciliary activity and caused necrosis of the epithelium. Mycoplasma granularum and M. hyosynoviae caused cessation of ciliary movement, but to a less extent, and necrosis did not occur. With M. hyopneumoniae , the infected organ cultures survived longer than the control cultures.     

Mycoplasma hyosynoviae Isolation from the Upper Respiratory Tract and Tonsils of Pigs

The occurrence of Mycoplasma hyosynoviae at different locations of the upper respiratory tract and tonsils of pigs was investigated in herds with problems of arthritis apparently caused by this microorganism. The isolation of M. hyosynoviae was facilitated by the use of a medium selectively suppressing the growth of Mycoplasma hyorhinis. M. hyosynoviae was cultured from 106 of 178 tonsils of slaughterhouse pigs from 8 herds but could not be isolated from the mucosa of the nasal cavity or the oral-pharyngeal area of 100 living, 10–20 weeks old pigs in 5 of the herds. The value of the selective principles in the medium appears from the circumstance that 86 of the 106 isolates were obtained despite the presence of M. hyorhinis. It is concluded that the tonsil is a reservoir for M. hyosynoviae and is probably the location of choice for an easy demonstration of the presence of this mycoplasma in a pig herd.     

Hydrogen metabolism of Azospirillum brasilense in nitrogen-free medium

Production of H2 by Azospirillum brasilense under N2-fixing conditions was studied in continuous and batch cultures. Net H2 production was consistently observed only when the gas phase contained CO. Nitrogenase activity (C2H2 reduction) and H2 evolution (in the presence of 5% CO) showed a similar response to O2 and were highest at 0.75% dissolved O2. Uptake hydrogenase activity, ranging from 0.3 to 2.5 mumol H2/mg protein per hour was observed in batch cultures under N2. Such rates were more than sufficient to recycle nitrogenase-produced H2. Tritium-exchange assay showed that H2 uptake was higher under Ar than under N2. Uptake hydrogenase was strongly inhibited by CO and C2H2. Cyclic GMP inhibited both nitrogenase and uptake hydrogenase activities.     

Energetic analysis of the growth of Methanobrevibacter arboriphilus A2 in hydrogen-limited continuous cultures

Hydrogen-limited chemostat cultures of Methanobrevibacter arboriphilus A2 were carried out. The available electron balance and carbon balance in M. arboriphilus A2 and other methanogenic strains grown on various substrates were well satisfied. This indicates that no extracellular organic products were formed during methanogenic growth. The molar growth yields for methane (Y(X/CH(4) )) were calculated as 1.06-1.42 g cell/mol CH(4) at dilution rate (0.21-0.43 day(-1)). The smaller Y(X/CH(4) ) of M. arboriphilus A2 compared with that of the other methanogenic strains was probably owing to the low growth rate of M. arboriphilus A2. The low value of Y(X/CH(4) ) may be favorable for methane fermentation because less sludge accumulation is expected. The efficiency of free energy transduction to ATP during methane formation from H(2) + CO(2) was 12-17% at the dilution rate (0.21-0.43 day(-1)) assuming that Y(ATP) was 6.5 g/mol and the free energy change of CO(2) reduction to methane with H(2) was -62.8 kJ/mol under physiological conditions.     

A strain of Melissococcus pluton cultivable on chemically defined media

Abstract A Brazilian strain of Melissococcus pluton multiplied well under anaerobic conditions on chemically defined media buffered with potassium phosphate. Thymine, xanthine, pyridoxal and one or more other vitamins, CO₂, glucose, methionine and possibly other amino acids, were essential. Other strains multiplied only slightly and could not be subcultivated on the media. The Brazil strain is the least related serologically to all known strains of M. pluton and is the least fastiduous when cultivated on other media. It has a DNA base composition of 31.4 G + C mol%, whereas others tested have been 29.0 and 30.2 G + C mol%.     

Factors influencing microbiological assay of cell-culture mycoplasmas

Summary A total of 6432 cell cultures was assayed for mycoplasmas over a 6-year period by aerobic and anaerobic incubation of agar and broth media. Mycoplasmas were detected in 375 cultures (5.8%).M. orale andA. laidlawii accounted for 61.3% of the isolates. Anaerobic incubation detected 98.1% of the isolates; aerobic incubation detected 45.8%. Of factors studied to determine their effect on mycoplasma assay, only two, anaerobic incubation and presence of mycoplasmacidal/static antibiotics, were significant. In separate studies, 86 of 2656 cell cultures (3.2%) were infected with strains ofM. hyorhinis that did not grow on cell-free media. Recommendations are given for microbiological assay of cell-culture mycoplasmas. These studies were supported in part by Contracts N01-AG-4-2865 and N01-AG-8-2117 from the National Institute on Aging and N01-GM-6-2119 from the National Institute of General Medical Sciences.     

Phospholipase A and glycerophosphodiesterase activities in the cell membrane of Mycoplasma hyorhinis

Mycoplasma hyorhinis, the major contaminant of tissue cultures, has been implicated in a variety of diseases in swine. Most human and animal mycoplasmas remain attached to the surface of epithelial cells. Nonetheless, we have recently shown that M. hyorhinis is able to invade and survive within nonphagocytic melanoma cells. The invasion process may require the damaging of the host cell membrane by either chemical, physical or enzymatic means. In this study, we show that M. hyorhinis membranes possess a nonspecific phospholipase A (PLA) activity capable of hydrolyzing both position 1 and position 2 of 1-acyl-2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] aminododecanoyl) phosphatidylcholine. In silico analysis of the M. hyorhinis genome shows that the PLA of M. hyorhinis shares no homology to described phospholipases. The PLA activity of M. hyorhinis was neither stimulated by Ca (2+) nor inhibited by EGTA and had a broad pH spectrum. Mycoplasma hyorhinis also possess a potent glycerophosphodiesterase (GPD), which apparently cleaves the glycerophosphodiester formed by PLA to yield glycerol-3-phosphate. Possible roles of PLA and GPD in invading host eukaryotic cells and in forming mediators upon the interaction of M. hyorhinis with eukaryotic cells are suggested.