Growth and removal of nitrogen and phosphorus by free-living and chitosan-immobilized cells of the marine cyanobacterium Synechococcus elongatus

This study investigated the growth rate of chitosan-immobilized cells of the marine cyanobacterium Synechococcus elongatus and its potential application in the removal of nitrogen and phosphorus for wastewater treatment. Immobilized cell cultures had a lag phase of growth due to the immobilization method, and their growth rate was similar to that of free-living cell cultures. Ammonia removal was higher in free cells (54%) than in immobilized cells (29%), but nitrate removal was similar in immobilized (38%) and free cells (44%); phosphorus removal was more efficient in free cells (88%) than in immobilized cells (77%). Chlorophyll a and protein content were higher in immobilized cells. Our study demonstrates that S. elongatus immobilized into chitosan capsules can remove nutrients and is able to maintain a growth rate comparable to that of free cells in culture.     

Helicobacter pylori Induces Apoptosis of T- and B-Cell Lines and Translocates Mitochondrial Apoptosis-Inducing Factor to Nucleus

Immune cell apoptosis may play a role in human persistent Helicobacter pylori infection. We planned to study the apoptosis of T and B cells by H. pylori strains. T (Jurkat) and B (Raji) cell lines were co-cultured with cagA-positive H. pylori strains carrying different vacA genotypes (s1a/m1, s1a/m2, and s2/m2). Apoptosis was detected by microscopy, DNA fragmentation assay, and flow cytometry. Apoptosis-inducing factor (AIF) transfer from mitochondria to nucleus was studied by immunoblot analysis. Apoptosis of T and B cells was significantly higher in H. pylori-infected cells than in uninfected controls (s1a/m1 80%, s1a/m2 78%, s2m2 69% vs. control 16% for T cells, P < 0.001; s1 a/m1 78%, s1a/m2 73%, s2m2 62% vs. control 24% for B cells, P < 0.001 by flow cytometry) with no difference among the genotypes. AIF transfer from mitochondria to nucleus was demonstrated in both apoptotic cell lines. Thus, H. pylori induces apoptosis in T- and B-cell lines and translocates AIF. T and B cells deletion through apoptosis may explain the persistence of H. pylori infection; its role in pathogenesis needs further research.     

Investigation of saturated and aromatic hydrocarbon resistance mechanisms in Pseudomonas aeruginosa IBBML1

Pseudomonas aeruginosa IBB_ML1, isolated from Poeni petroleum sludge, was able to tolerate and degrade both saturated (n-hexane, n-heptane, n-hexadecane, cyclohexane) and aromatic (benzene, ethylbenzene, propylbenzene, xylene isomers, styrene) hydrocarbons. Molecular studies have revealed that the high hydrocarbon resistance of Pseudomonas aeruginosa IBB_ML1 could be due to the action of members of the HAE1 (hydrophobe/amphiphile efflux 1) family of transporters. It is further possible that additional mechanisms may account for the tolerance of Pseudomonas aeruginosa IBB_ML1 to hydrocarbons, and a combination of short-term and long-term mechanisms may act together in the adaptation of Pseudomonas aeruginosa IBB_ML1 cells to saturated and aromatic hydrocarbons. β-galactosidase activity measurements revealed that there was significant induction of the lacZ gene in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of either 5% and 10% (v/v) saturated or aromatic hydrocarbons, compared with control (cells incubated without hydrocarbons). Rhodamine 6G accumulation in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of 5% and 10% (v/v) saturated hydrocarbons was higher than rhodamine 6G accumulation in cells grown in the presence of 5% and 10% (v/v) aromatic hydrocarbons. The study of cellular and molecular modifications to Pseudomonas aeruginosa IBB_ML1 induced by 5% and 10% (v/v) saturated and aromatic hydrocarbons revealed a complex response of bacterial cells to the presence of different hydrophobic substrates in the culture medium.     

The thyroid C cells of ovariectomized rats treated with estradiol

The structure and function of thyroid C cells were studied in ovariectomized (Ovx) adult female rats without and after chronic treatment with estradiol dipropionate (EDP). A peroxidase–antiperoxidase method was applied for localization of calcitonin (CT) in the C cells. Morphometric changes in their volume, nuclei, and relative volume density were evaluated in comparison with sham-operated control rats using a stereological method. The number of C cells was calculated. CT content in the sera was determined by radioimmunoassay. Ovariectomy (Ovx) led to a 21% increase in body weight (P<0.005), while treatment of Ovx rats with EDP decreased body weight by 25% (P<0.01). The immunoreactivity for CT in C cells of the Ovx rats was markedly increased. Significant decreases in the volume of C cells (by 13%; P<0.05) and serum CT (by 45%) were recorded, while the C cell number increased by 59% (P<0.05) in relation to the corresponding controls. The treatment of Ovx rats with EDP caused conspicuous degranulation of the C cells. The cellular volume was increased by 11% and serum CT by 36% in comparison with Ovx animals. At the same time a decrease in C cell number by 29% (P<0.05) was evident. It may be concluded that estradiol deficiency after Ovx reduced the synthesis and release of CT, while chronic treatment of these animals with EDP had a positive effect on the secretory activity of thyroid C cells.     

Immunization of mice with cells from juvenile worms of <Emphasis Type="Italic">Schistosoma japonicum</Emphasis> provides immunoprotection against schistosomiasis

To validate the protective efficacy against schistosomiasis by immunization with cells from juvenile Schistosoma japonicum in a murine model and to analyze possible factors related to protection, in this study, two independent repeated vaccination trials were performed. After three subcutaneous vaccinations, in trial one, in the absence of adjuvant, primary juvenile worm cells (pJCs) from S. japonicum induced remarkable average reductions in worm burden (54.3%), liver eggs per gram (LEPG) load (59.8%) as well as egg granulomas size (66.5%) compared to PBS control group (P<0.01), which were significantly higher than those elicited by fractions of juvenile worm cells (JCFs) or fractions of juvenile worms (JWFs) (P<0.05). Non-cell components of worms (WNCs) showed no significant protection. In trial two, compared to PBS control group, significant protective effect was also observed for cultured juvenile worm cells (cJCs) from S. japonicum with 58.4% worm reduction and 68.1% LEPG reduction (P<0.01). However, cultured adult worms cells (cACs) showed significantly higher worm burden (P<0.05) and egg burden (P<0.01) when compared to cJCs. Immunological analysis of trial two revealed that cJCs engendered a Th1-biased mixed Th1/Th2 type of immune response while cACs elicited a Th2-type response. Our data indicated that immunization with both primary and cultured cells from S. japonicum juvenile worms provided high immunoprotection, for which the physical character of immunogens, stage-specific parasite and the type of immune response induced might be responsible, suggesting that vaccination with whole cells from S. japonicum larvae is a promising approach to produce protective immunity against schistosomiasis.     

Contributions of immobilized and free cells of salt-tolerantZygosaccharomyces rouxii andCandida versatilis to the production of ethanol and 4-ethylguaiacol

Summary The contribution of immobilized cells and free cells released from gel beads to ethanol production by the salt-tolerant yeastsZygosaccharomyces rouxii andCandida versatilis, and 4-ethylguaiacol (4-EG) production byC. versatilis were investigated using an airlift reactor. The amounts of ethanol produced by free cells were about 65% and about 90% of total ethanol in the reactor forZ. rouxii andC. versatilis, respectively. It was found that immobilized cells gave a much lower specific productivity of ethanol (ethanol production per hour per cell) than free cells of both yeasts, especially ofC. versatilis. 4-EG was produced mainly by immobilized cells ofC. versatilis; the amount of 4-EG produced by free cells was about 20% of the total 4-EG, in contrast to the results of ethanol production. However, the specific productivity of 4-EG (4-EG production per hour per cell) by immobilized and free cells was fairly similar.     

A comparison of the reproductive ability of <Emphasis Type="Italic">Varroa destructor</Emphasis> (Mesostigmata:Varroidae) in worker and drone brood of Africanized honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Colony infestation by the parasitic mite, Varroa destructor is one of the most serious problems for beekeeping worldwide. In order to reproduce varroa females, enter worker or drone brood shortly before the cell is sealed. To test the hypothesis that, due to the preference of mites to invade drone brood to reproduce, a high proportion of the mite reproduction should occur in drone cells, a comparative study of mite reproductive rate in worker and drone brood of Africanized honey bees (AHB) was done for 370 mites. After determining the number, developmental stage and sex of the offspring in worker cells, the foundress female mite was immediately transferred into an uninfested drone cell. Mite fertility in single infested worker and drone brood cells was 76.5 and 79.3%, respectively. There was no difference between the groups (X ² = 0.78, P = 0.37). However, one of the most significant differences in mite reproduction was the higher percentage of mites producing viable offspring (cells that contain one live adult male and at least one adult female mite) in drone cells (38.1%) compared to worker cells (13.8%) (X ² = 55.4, P < 0.01). Furthermore, a high level of immature offspring occurred in worker cells and not in drone cells (X ² = 69, P < 0.01). Although no differences were found in the percentage of non-reproducing mites, more than 74% (n = 85) of the mites that did not reproduce in worker brood, produced offspring when they were transferred to drone brood.     

Effect of cultivation time and growth medium on the coaggregation capability of <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> S35

The effects of different cultivation periods and growth media on the cell surface hydrophobicity and coaggregation ability of Acinetobacter johnsonii S35 with Oligotropha carboxidovorans S23 (both sewage sludge isolates) were examined. It was observed that when cultivated in polypeptone medium, A. johnsonii S35 shows high hydrophobicity and strong coaggregation (98% aggregation index) with O. carboxidovorans S23, which remain unaltered after a 1-day, 3-day or 7-day cultivation period. When A. johnsonii S35 was cultivated with different carbon substrates, large coaggregates and a high aggregation index were observed using acetate-grown cells (96%), followed by ethanol-(83%) and vegetable oil-grown cells (72%). Coaggregates were small and narrow in ethanol-grown cells and still smaller in case of the vegetable oil-grown cells. Although no direct correlation could be observed, more hydrophobic cells showed a higher aggregation index. None of the culture supernatants of A. johnsonii S35 showed emulsification of hexadecane or a significant ability to aggregate O. carboxidovorans S23 cells, indicating that the coaggregation factor of A. johnsonii S35 is cell-bound. These results demonstrate that the coaggregation ability of A. johnsonii S35 is constitutive and is not lost under diverse cultivation conditions.     

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO2

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO₂ concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1–4 days. In contrast, C. littorale cells which had been grown in 5% CO₂ (5% CO₂-grown cells) could grow in 40% CO₂ without any lag period. When air-grown cells were transferred to 40% CO₂, the quantum efficiency of PS II (Φ_II) decreased greatly, while no decrease in Φ_II was apparent when the 5% CO₂-grown cells were transferred to 40% CO₂. In contrast to air-grown cells, 5% CO₂-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO₂-grown cells to air, photosynthetic susceptibility to 40% CO₂ was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO₂. The intracellular pH value (pH_i) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO₂ for 1–2 h, but no such decrease in pH_i was apparent in the presence of EZA. Both air- and 5% CO₂-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO₂ concentrations, grew in 40% CO₂ without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO₂. No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sulfhydryl oxidase immunoreactivity in seminiferous tubules of infertile men

Summary Sulfhydryl oxidase (SOx) immunoreactivity was investigated in the seminiferous epithelium of human biopsy material from the testes of 33 adult men with disturbed fertility. SOx immunoreactivity was expressed in normal seminiferous epithelium in type-A spermatogonia (27±4% of all spermatogonia) (n=4), in spermatocytes and round spermatids. Mature spermatozoa as well as Sertoli cells were unlabelled. within the interstitium, Leydig cells were immunopositive. In biopsies of oligozoospermic men showing hypospermatogenesis (n=24), an increase in labelled spermatogonia up to more than 90% was observed in biopsies, where seminiferous epithelia revealed only spermatogonia and Sertoli cells. Within the group of oligozoospermic patients there was a significant increase of labelled spermatogonia from 43±13% (>20 mill/ejaculate) (n=7) to 55±16% ( 20 and >20 mill/ejaculate) (n=6) to 68±8% (<5 mill/ejaculate) (n=11) and a significant (P=0.01) decrease of score count from 7.0±2.7 to 2.0±1.8. In this group the increase of labelled spermatogonia was correlated with sperm concentrations in the ajaculate (correlation coefficient: r=-0.6). In biopsies of azoospermic patients showing maturation arrest at the level of spermatocytes or spermatids (n=5) the percentage of labelled spermatogonia was within the range of 24% to 59%. Immunoreactivity in Sertoli cells was only found in single degenerating cells and in tubules showing Sertoli Cell Only Syndrome (SCO) without lumen formation. Sertoli cells within immature seminiferous cords were immunonegative, indicating that Sertoli cell SOx immunoreactivity is rather a sign of physiological alterations in degenerating cells than dependent on the stage of differentiation. Leydig cells did not show changes of immunoreactivity in any biopsy. It is concluded that SOx expression in spermatogonia may serve as a marker for spermatogenic efficiency.     

Stimulation of the side population fraction of ATDC5 chondroprogenitors by hypoxia

The influence of oxygen tension on the side population (SP) fraction sorted from ATDC5 chondroprogenitor cells was investigated. ATDC5 cells cultured in normoxia (20%) or hypoxia (1% oxygen) were compared. The SP fraction was significantly higher in the cells cultured in hypoxia. The gene expression of 3 ABC transporters, abcb1a/b (mdr1a/b) and abcg2 (bcrp1) was quantified by RT-PCR. SP cells were characterized by the higher expression of abcb1a. The expression levels of abcb1b and abcg2 were higher than abcb1a. However, there was no significant difference between SP and non-SP fractions in the expression of abcb1b and abcg2. The telomeric repeat amplification protocol assay showed that SP cells tended to show lower telomerase activity than non-SP cells. Chondrogenic properties of ATDC5 cells derived from SP or non-SP were assessed by micromass culture. There were not significant differences between SP and non-SP derived cells in Alcian blue staining and sox9, Aggrecan, Col2a1 and SZP mRNA expression. The results demonstrate that the SP fraction was stimulated by hypoxia and chondrogenic properties of SP and non-SP fraction of ATDC5 cells were similar.     

M and N proteins of SARS coronavirus induce apoptosis in HPF cells

Background: SARS-associated coronavirus (SARS-CoV) induced cell apoptosis and its structural proteins may play a role in this process. Objectives: To determine whether the structural proteins M and N of SARS-CoV induce apoptosis. Study design: We investigated human pulmonary fibroblast (HPF) cells, were transfected with plasmids containing the M or N gene, by TdT-mediated dUTP nick end labeling (TUNEL), Hoechst 33342 staining for nuclei, and observation of morphology. Results: We found that in the absence of serum about 16.34% of cells transfected by pcDNA3.1-M and 21.72% of N-transfected cells showed typical apoptotic characteristics, significantly different from mock-transfected cells (only 6.23%, p<0.01). Furthermore, the cells that were co-transfected with M and N proteins showed more obvious phenomena of cell death (about 36.03%). There was a statistical significance between M-transfected cells and co-transfected cells (p<0.01), and a remarkable difference between N-transfected cells and co-transfected cells (p<0.01). Conclusions: The results show that M and N proteins of SARS-CoV can induce apoptosis of HPF cells. Co-transfection of M and N enhances the induction of apoptosis by M or N alone, which also suggests that the structural proteins of SARS-CoV may play an important role not only in the process of invasion but also in the pathogenetic process in cells.     

Use of isotopically chiral [4′-13C]penciclovir and 13C NMR to determine the specificity and absolute configuration of penciclovir phosphate esters formed in HSV-1- and HSV-2-infected cells and by HSV-1-encoded thymidine kinase

Penciclovir is a potent antiherpesvirus agent which is highly selective due to its phosphorylation only in virus infected cells. Phosphorylation of one of the hydroxymethyl groups of penciclovir (PCV) creates a chiral centre leading to the possible formation of (R)- and (S)-enantiomers. The absolute configuration and stereospecificity of the PCV-phosphates produced in cells infected with herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), as well as by HSV-1-encoded thymidine kinase, were determined using isotopically chiral [4′-¹³C]PCV precursors and ¹³C NMR spectroscopy of the isolated metabolites. The absolute configuration of penciclovir-triphosphate (PCV-TP) produced in HSV-1-infected cells was shown to be S with an enantiomeric purity of greater than 95%. However, in contrast to HSV-1-infected cells in which none of the (R) enantiomer was detected, about 10% of (R)-PCV-TP was produced in HSV-2-infected cells. Phosphorylation of PCV by HSV-1-encoded thymidine kinase was found to give 75% (S)- and 25% (R)-PCV-monophosphate. The proportion of the (S)-isomer appears to be amplified in the subsequent phosphorylations leading to the triphosphate. © 1993 Wiley-Liss, Inc.     

Photorespiratory ammonium assimilation in chloroplasts of Chlamydomonas reinhardtii

The effect of CO₂ concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw-15 mutant (lacking cell wall) and in the high CO₂-requiring double mutant cia-3/cw-15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw-15 cells, both the extracellular (CA_ext) and chloroplastic (CA_chl) CA activities increased after transferring cells from media bubbled with 5% CO₂ in air (v/v, high-C_i cells) to 0.03% CO₂ (low-C_i cells), whereas in cia-3/cw-15 cells only the CA_ext was induced after adaptation to low-C_i conditions and the CA_chl activity was negligible. During adaptation to low-C_i conditions in the presence of 1 mM of l-methionine-D,L-sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw-15 in the presence of MSX was lower in cells grown and kept at 5% CO₂ than in high-C_i cells adapted to 0.03% CO₂. The double mutant cia-3/cw-15 excreted photorespiratory ammonium at a higher rate than did cw-15. Total GS activity (GS-1 plus GS-2) increased during adaptation to 0.03% CO₂ in both strains of C. reinhardtii. However, only the activity GS-2, which is located in the chloroplast, increased during the adaptation to low CO₂, whereas the cytosolic GS-1 levels remained similar in high and low-C_i cells. We conclude that: (1) cia-3/cw-15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS-2/GOGAT cycle; and (3) chloroplastic GS-2 concentration changes in response to the variation of environmental CO₂ concentration.     

Differences in protein patterns of gill epithelial cells of the fish Gillichthys mirabilis after osmotic and thermal acclimation

Different protein patterns in gill epithelium of a euryhaline and eurythermal teleost fish (Gillichthys mirabilis, Family Gobiidae) in response to long-term (2 months) osmotic and thermal acclimation were found for the first time. Gill epithelial cells were isolated to remove extracellular proteins and quantify specialized cell types. Chloride cells were identified on the basis of size (>10 m) and bright appearance after [2-(p-dimethylaminostyryl)-1-methyl-pyridinium-iodine] staining. Small mitochondria-rich cells were <5 m in diameter and showed intermediate fluorescence. Abundance of chloride cells and small mitochondria-rich cells was significantly influenced by osmotic but not thermal acclimation (dilute seawater/25°C: 1.4±0.2% chloride cells, 11.9±4.6% small mitochondria-rich cells; seawater/25°C: 2.4±0.6% chloride cells, 2.2±1.3% small mitochondria-rich cells; seawater/10°C: 2.9±0.3% chloride cells, 1.2±0.7% small mitochondria-rich cells). Pavement cells, identified by low fluorescence and intermediate size (5–10 m), largely predominated under all conditions (>85% of cells). Thus, they represented the major protein source in gill epithelium. Differences in protein patterns were detectable using two-dimensional but not one-dimensional electrophoresis. Of 602 proteins identified by charge and molecular weight properties, only two were induced by high temperature (25°C) and three in response to cold acclimation (10°C). Nine proteins were induced in diluted seawater-acclimated fish, whereas no seawater-induced proteins were found. We hypothesize that proteins induced under dilute seawater conditions are important for the function of pavement cells in gills of hyper-osmoregulating G. mirabilis.Abbreviations BCA bicinchoninic acid - BSS balanced salt solution - CC chloride cells - CLB cell lysis buffer - DASPMI [2-(p-dimethylaminostryryl)-1-methylpyridinium-iodine] - DSW diluted sea water - DTT dithiothreitol - EDTA ethylene-diaminetetraacetate - FW fresh water - IEF isoelectric focusing - PC pavement cells - PDA diacrylpiperazine - pI isoelectric point(s) - PMSF phenylmethanesulphonylfluoride - SDS sodium dodecyl sulfate - SMRC small mitochondria-rich cells - SW sea water - TEMED tetramethylenediamine     

Diel Division Cycle and Growth Rates of Synechococcus in Lakes Huron and Michigan1

A clone of Synechococcus isolated from Lake Huron and natural populations of Synechococcus from lakes Huron and Michigan were studied in 1989 to examine the diel division cycle and to provide estimates of the in situ growth rate based on the frequency of dividing cells (FDC) method. Cultured populations of Synechococcus exhibited a consistent diel division pattern with a midday/afternoon (1100–1800 h) peak in the percent of dividing cells. The maximum percent of dividing cells varied among cultures (8-27%) and was related to the growth rate. A small fraction of dividing cells (3-5%) remained throughout the dark period, suggesting that some cells were arrested in the doublet stage prior to division. The duration of division (t_d) ranged from 2.6-4.9 h, with a 3.7 h mean for cultures with growth rates ≥0.34 d⁻¹ but increased to 8 h at a lower growth rate of 0.20 d⁻¹. The diel division pattern for natural populations was very similar to the laboratory clone; an afternoon peak (1400-2100 h) in dividing cells and a small fraction of dividing cells (2-5%) remained during the dark period. The maximum percent of dividing cells for natural populations ranged from 6-10%. In situ growth rates, determined from the FDC and assuming a constant t_d of 3.7 h, ranged from 0.30-0.42 d⁻¹. The FDC method may provide accurate estimates of in situ growth, particularly in environments where the growth rate is >0.34 d⁻¹, but in lakes Huron and Michigan where growth rates can be lower and t_d values may increase, FDC-growth rates must be viewed with caution.     

Utilization of Proteinase K-Treated Cells as Lipopolysaccharide Antigens for the Serodiagnosis of Helicobacter pylori Infections

We have evaluated the use of proteinase K (PK)-treated cells isolated from Helicobacter pylori as lipopolysaccharide (LPS) antigens in an immunoblot assay and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of H. pylori infection. The sera from patients with chronic gastritis, gastric ulcer, duodenal ulcer or gastric cancer, and from healthy adults with or without H. pylori infection were assayed with three commercial serodiagnostic kits (HM-CAP, Helico-G, and G.A.P. II) and novel methods relying on the use of PK-treated cells. The PK-treated cells used in these assays were selected on the basis of their possibility to possess a common epitope in the O-polysaccharides of H. pylori, which is known to be highly immunogenic in humans. Of the sera from these patients, 71-94% were positive with the commercial kits, 97% with immunoblot assay, and 90% with ELISA. On the other hand, of the healthy adults infected with H. pylori, 72-97% were positive with the commercial kits, 86% with immunoblot assay, and 72% with ELISA. PK-treated cells that did not contain the common epitope were unsuitable as an antigen for immunoblot assay or ELISA. Furthermore, the reactivity of these sera reacted specifically with H. pylori PK-treated cells but not with LPSs from other gram-negative bacteria, such as Campylobacter, Proteus, Bordetella, and Salmonella. These results demonstrate that the serological assays relying on the use of H. pylori PK-treated cells possessing a highly antigenic epitope are potentially useful as a serodiagnostic test for H. pylori infection.     

Dedifferentiation derived cells exhibit phenotypic and functional characteristics of epidermal stem cells

Differentiated epidermal cells can dedifferentiate into stem cells or stem cell‐like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation‐derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c‐nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and β₁‐integrin emerged at the wound‐neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and β₁‐integrin⁺ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10⁺ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation‐derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, β₁‐integrin, Oct4 and Nanog in dedifferentiation‐derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation‐derived cells demonstrated: (1) the numbers of colonies consisting of 5–10 cells and greater than 10 cells were increased 5.9‐fold and 6.7‐fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 × 10⁵ cells versus 1 × 10¹² cells) were all significantly increased and (4) dedifferentiation‐derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation‐derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.     

Secretion rates and levels of vascular endothelial growth factor in clone A or HCT-8 human colon tumour cells as a function of oxygen concentration

Molecular and in situ hybridization studies have shown, in a number of cell types, that under hypoxic conditions, vascular endothelial growth factor (VEGF) mRNA expression is up-regulated and VEGF protein is concomitantly increased. To establish a quantitative relationship between VEGF protein levels and oxygenation, we exposed exponentially growing clone A or HCT-8 human colon tumour cells in vitro (22 h at 37°C) to oxygen concentrations from 21% (air mixture) to 0.01%. Protein levels in cells and medium were then assayed using an enzyme-linked immunoabsorbent assay (ELISA). Intracellular levels of VEGF in clone A or HCT-8 cells exposed to either air (21% O₂) or the 0.01% O₂ mixture respectively increased from about 73 to 1270, and 1.5 to 1180 pg/10⁶ cells (about 17- and 80-fold increases). The shapes of the response curves (log of the intracellular VEGF concentrations v. log oxygen concentration) for both cell types were sigmoidal. However, intracellular VEGF levels in HCT-8 cells were always less than that of clone A cells until levels of about 0.3 to 0.1% O₂ were reached. Levels of VEGF in the supernatant were also increased after the 22 h hypoxic exposures. Because cell proliferation and clonogenicity were also measured, it was possible to estimate the secretion rates of VEGF for both cell lines as a function of oxygen percentage. For clone A cells, the secretion rate (pg/10⁶ cells/h) in 21% O₂ was 62.5. This rate increased to 428.8 pg/10⁶ cells/h at 0.01% O₂, a 7-fold increase. For HCT-8 cells, levels in the medium at 21% O₂ were too low to be measured by ELISA. However, between 10% and 0.01% O₂, secretion rates increased from 5.0 to 376.0 pg/10⁶ cells/h, a 75-fold increase. Therefore, at very low O₂ levels, VEGF secretion rates were similar in the two cell lines. We propose that the different VEGF responses of clone A and HCT-8 colon tumour cells to hypoxic stress in vitro are related to the in vivo observation that the respective hypoxic percentages of solid neoplasms originating from these cell lines are markedly different (i.e. about 3 versus 80%) at equivalent volumes of 750 mm³.     

Naphthoquinone contents of in vitro cultured plants and cell suspensions of Dionaea muscipula and Drosera species

In vitro cultured carnivorous plants were grown on a hormone-free medium. They produced the following naphthoquinones: Dionaea muscipula (plumbagin: 5.3%), Drosera rotundifolia (7-methyljuglone: 0.6%), D. binata (plumbagin: 1.4%), and D. capensis (7-methyljuglone: 0.5%). A red, slow-growing suspension culture of D. muscipula was maintained in a modified McCowns Woody Plant (McC) medium and produced plumbagin (2.59%) after 30 days growth. A suspension culture of D. rotundifolia grew slowly as multicoloured small aggregates only in a modified Murashige and Skoog (MS) medium. No quantifiable amounts of naphthoquinones were produced. Several cell lines of D. capensis were developed. Green aggregates grown in a modified MS medium contained 7-methyljuglone (0.33%) and differentiated into plants when placed onto hormone-free medium. Pink cultures grown in modified McC medium contained 7-methyljuglone (1.24%), while dark red cultures produced ca. 1% in both modified McC and MS media. Though the latter medium was significantly better with regard to biomass production, cells excreted a mucin when cultured in both media (0.21 g dry mucin/g dry cells in McC) and (0.16 g dry mucin/g dry cells in MS). Effects of the presence or absence of light during the growth period of 30 days showed that there was no effect on biomass and only slight effects on mucin production and naphthoquinone contents.