Retention of basic electrophysiologic properties by human sweat duct cells in primary culture

Summary The present investigation was undertaken to examine the usefulness of cultured human sweat duct cells for ion transport and related studies in the genetic disease, cystic fibrosis. Electrical properties of cultured duct (CD) cells were compared with electrical properties of microperfused duct (MPD) cells. The resting apical membrane potential (V _a ) of the CD cells was −26.4±0.9 mV,n=158 cells as compared to −24.3±0.6 mV,n=105 of MPD cells. The Na⁺−K⁺ pump inhibitor ouabain, when applied to the apical surface of the CD cells and basolateral surface of MPD cells, depolarized both CD cells (from −28.6±3.6 to −16.8±2.4 mV,n=5) and MPD cells (from −23.8±0.5 mV to −19.5±1.8 mV,n=6). The Na⁺ conductance inhibitor amiloride applied to the apical surface hyperpolarized the apical membrane potentials (V_a) of CD cells and MPD cells by −13.2±1.4 mV,n=43 and −34.3±3.1 mV,n=19), respectively, indicating the presence of amiloride sensitive Na⁺ channels in both groups of cells. However, the amiloride sensitivity of CD cells was dependent on the age of the culture. Cl⁻ substitution at the apical side by the impermeant anion gluconate depolarized the V _a of CD cells and MPD cells by 12.2±0.9 mV,n=32 and 37.9±4.3 mV,n=12, respectively. The effect of β-adrenergic agonist isoproterenol (IPR), was inconsistent. In CD cells, IPR either hyperpolarized (ΔV _a =−8.3±1.2mV,n=5) or depolarized (ΔV _a =8.2±2.3 mV,n=4) or had no effect,n=2. In contrast, most of the MPD cells did not respond to IPR, but three cells had a varied response to IPR. Our results suggest that CD cells, like MPD cells, retain significant Na⁺ and Cl⁻ conductances. CD cells seem to have developed a higher sensitivity to β-adrenergic stimulation in tissue culture as compared to MPD cells. This work was supported by grants from the National Institutes of Health, Bethesda, MD, DK26547, Getty Oil Co., the Gillette Co., Cystic Fibrosis Research Inc., and the U.S. National Cystic Fibrosis Foundation.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment

Objective Chronic inflammation and cancer development are associated with dysregulated immune responses and the presence of regulatory T cells (T_reg). To study the role of T_reg in tumor cell escape from immune surveillance, an in vitro model simulating the tumor microenvironment and promoting the induction and expansion of IL-10⁺ T_reg type 1 (Tr1) was established. Methods An in vitro co-culture system (IVA) included an irradiated head and neck squamous cell carcinoma cell line, immature dendritic cells (iDC), CD4⁺CD25⁻ T cells and cytokines, IL-2 (10 IU/ml), IL-10 (20 IU/ml), IL-15 (20 IU/ml) ± 1 nM rapamycin. Autologous iDC and CD4⁺CD25⁻ T cells were obtained from the peripheral blood of 15 normal donors. Co-cultures were expanded for 10 days. Proliferating lymphocytes were phenotyped by multi-color flow cytometry. Their suppressor function was measured in CFSE inhibition assays ± neutralizing anti-IL-10 mAb and using transwell cultures. Culture supernatants were tested for IL-4, IL-10, TGF-β and IFN-γ in ELISA. Results In the IVA, low doses of IL-2, IL-10 and IL-15 promoted induction and expansion of CD3⁺CD4⁺CD25⁻IL2Rβ⁺IL2Rγ⁺FoxP3⁺CTLA-4⁺IL-10⁺ cells with suppressor activity (mean suppression ± SD = 58 ± 12%). These suppressor cells produced IL-10 (mean ± SD = 535 ± 12 pg/ml) and TGF-β (mean ± SD = 512 ± 38 pg/ml), but no IL-4 or IFN-γ. Suppressor function of co-cultures correlated with the percent of expanding IL-10⁺ Tr1 cells (r ² = 0.9; P < 0.001). The addition of rapamycin enriched Tr1 cells in all co-cultures. Neutralizing anti-IL-10 mAb abolished suppressive activity. Suppression was cell-contact independent. Conclusion The tumor microenvironment promotes generation of Tr1 cells which have the phenotype distinct from that of CD4⁺CD25^highFoxP3⁺ nTreg and mediate IL-10 dependent immune suppression in a cell-contact independent manner. Tr1 cells may play a critical role in cancer progression.     

Transglutaminase 2 silencing reduced the beta-amyloid-effects on the activation of human THP-1 cells

The aberrant expression and activation of transglutaminase 2 (TG2), the ubiquitous enzyme which catalyzes calcium-dependent protein cross-linking reactions, has been reported in many inflammatory diseases. Chronic inflammation, mediated by prolonged activation of brain-resident immunocompetent cells, appears to be involved in the pathogenesis of several age-related diseases, such as Alzheimer’s disease. Given that increased TG2 expression has been observed in AD brains, this study was aimed to characterize the role of TG2 in THP-1 monocytes stimulated with amyloid-beta (Aβ). Aβ_1–42 treatment dose-dependently increased TG2 expression in THP-1 cells. In particular, a fivefold up-regulation of TG2, compared with control cells, was observed in the presence of 0.5 μM Aβ_1–42. At the same concentration, Aβ_1–42 was able to promote monocyte maturation as suggested by increased expression of the cell surface antigen CD14 as well as the adhesion-promoting factor fibronectin. The stimulation of THP-1 cells with Aβ_1–42 also led to a significant up-regulation of tumor necrosis factor α (TNF-α) and matrix metalloproteinase 9 (MMP-9). Interestingly, THP-1 cell transfection with small interfering RNA directed against TG2 was able to reduce Aβ_1–42 increased levels of all the examined markers of monocyte maturation (CD14, fibronectin), and activation (TNF-α, MMP-9). These results indicate that TG2 up-regulation is required for the functional THP-1 monocyte activation induced by Aβ_1–42. This work suggests that TG2 inhibition may represent a therapeutic target to ameliorate the inflammation and progression in Alzheimer’s disease.     

Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche

In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a “biomimetic niche.” The CD34⁺ cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2–5 weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture, the CD34⁺ cells from umbilical cord blood in the 3D system increased 3.3–4.8 folds when compared with the initial CD34⁺ cells. CD34⁺/CD38⁻ cells accounted for 82–90% of CD34⁺ cells. After 5 weeks, CD34⁺/CD38⁻ cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 10³ vs. 1.0 ± 0.5 × 10⁴, p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 10³ vs. 2.5 ± 0.7 × 10², p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6–9.3 folds vs. 1.0–1.5 folds) after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2–7.2 folds increase over input at 2 weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.     

Postprandial acid–base balance and ion regulation in freshwater and seawater-acclimated European flounder, <Emphasis Type="Italic">Platichthys flesus</Emphasis>

The effects of feeding on both acid–base and ion exchange with the environment, and internal acid–base and ion balance, in freshwater and seawater-acclimated flounder were investigated. Following voluntary feeding on a meal of 2.5–5% body mass and subsequent gastric acid secretion, no systemic alkaline tide or respiratory compensation was observed in either group. Ammonia efflux rates more than doubled from 489 ± 35 and 555 ± 64 μmol kg⁻¹ h⁻¹ under control conditions to 1,228 ± 127 and 1,300 ± 154 μmol kg⁻¹ h⁻¹ post-feeding in freshwater and seawater-acclimated fish, respectively. Based on predictions of gastric acid secreted during digestion, we calculated net postprandial internal base gains (i.e., HCO₃⁻ secreted from gastric parietal cells into the blood) of 3.4 mmol kg⁻¹ in seawater and 9.1 mmol kg⁻¹ in freshwater-acclimated flounder. However, net fluxes of ammonia, titratable alkalinity, Na⁺ and Cl⁻ indicated that branchial Cl⁻/HCO₃⁻ and Na⁺/H⁺ exchange played minimal roles in counteracting these predicted base gains and cannot explain the absence of alkaline tide. Instead, intestinal Cl⁻/HCO₃⁻ exchange appears to be enhanced after feeding in both freshwater and seawater flounder. This implicates the intestine rather than the gills as a potential route of postprandial base excretion in fish, to compensate for gastric acid secretion.     

Confocal Raman microspectroscopy of the activation of single neutrophilic granulocytes

Confocal Raman micro-spectroscopy has been applied to investigate the activation process of single, living neutrophilic granulocytes. Both resting cells as well as activated cells were measured. The activation of cells was performed with phorbol-12-myristate-13-acetate activator and Escherichia Coli bacteria. Raman microspectroscopy combines a high spatial resolution inside a single, living cell with detailed material information. Using this approach it can be concluded that activation of the cells with phorbol-12-myristate-13-acetate causes a change in the redox state of cytochrome b₅₅₈. This protein is a part of the NADPH-oxidase complex that neutrophilic granulocytes employ to generate O₂ ^–, superoxide anion. Additionally a change in the redox state of myeloperoxidase can be observed. Myeloperoxidase is known to react with O₂ ^–. Activation of the cells with bacteria gives rise to corresponding changes in the Raman spectra. From this single cell study it can be concluded that the enzymes cytochrome b₅₅₈ and myeloperoxidase are present inside the cytoplasm of the living cell, while participating in the redox processes. Activation causes an intra-cellular release of oxygen metabolites. Activation with bacteria of neutrophilic granulocytes from a patient with chronic granulomatous disease, that contain no cytochrome b₅₅₈, led to typical changes in the redox state of myeloperoxidase. This indicates that in the bacterium/neutrophilic granulocyte system oxygen metabolites are generated that are capable of reacting with MPO. Received: 1 September 1998 / Revised version: 20 February 1998 / Accepted: 22 February 1998     

Growth pattern of <Emphasis Type="Italic">Bidens cernua</Emphasis> L.: relationships between relative growth rate and its physiological and morphological components

Seedlings of Bidens cernua L. emerged when mean air temperature was 17.0±1.3 °C. The highest net photosynthetic rate (P _N), 13.8±0.8 μmol(CO₂) m⁻² s⁻¹, was monitored during the vegetative period (May–August), decreasing on an average by 50 % during flowering (August–September) and during fruiting (September–November) phases. The senescence phase (October–November) was characterised by 79, 58, and 18 % decrease of P _N, chlorophyll content, and leaf area (LA), respectively, from the maximum values. The time span from seedling emergence to the end of fruiting phase was 202 d. The total plant biomass was 1.58±0.05 g of which 81 % was aboveground plant portion. The total dry mass relative growth rate averaged over the assimilation period was 0.0804±0.0002 kg kg⁻¹ d⁻¹, and it was correlated to both the net assimilation rate (NAR) and the leaf area ratio (LAR).     

Improved hydrogen photoproduction regulated by carbonylcyanide <Emphasis Type="Italic">m</Emphasis>-chlorophenylhrazone from marine green alga <Emphasis Type="Italic">Platymonas subcordiformis</Emphasis> grown in CO<Subscript>2</Subscript>-supplemented air bubble column bioreactor

To develop an integrated process of CO₂-fixation and H₂ photoproduction by marine green microalga Platymonas subcordiformis, the impact of algal cells grown in CO₂-supplemented air bubble column bioreactor was investigated on H₂ photoproduction regulated by carbonylcyanide m-chlorophenylhrazone. Highest cell growth (3.85 × 10⁶ cells ml⁻¹), starch content (0.25 ± 0.08 mg per 10⁶ cells) and hydrogen production (50 ± 3 ml l⁻¹) were achieved at 3% CO₂-supplemented culture, which are respectively 1.4, 2.1, 1.5-fold of the air-supplemented culture. Improved H₂ production correlated well with the increase in starch accumulation. In this process, the algal cells have been recycled for stable H₂ production of 40–50 ml l⁻¹ over five cycles.     

Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients

 We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR⁺ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3^–, CD19^–, CD20^–, CD14^–, CD11b^–, CD16^–, CD56^–). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean ± SE: 0.36 ± 0.05%, 0.14 ± 0.06%, and 0.75 ± 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system. Received: 20 June 1997 / Accepted: 14 August 1997     

Osmoregulation in juvenile and adult white sturgeon,Acipenser transmontanus

Synopsis Blood samples from cannulated young adult (2.5–15 kg) white sturgeon, acclimated to San Francisco Bay water (24 ppt) had plasma values of 248.8 ± 13.5 mOsm kg⁻¹ H₂O, [Na⁺] = 125 ± 8.0 mEq 1⁻¹, [K⁺] = 2.6 ± 0.8 mEq 1⁻¹ and [CL⁻] = 122 ± 3.0 mEq 1⁻¹. Freshwater acclimated sturgeon had an osmolality of 236 ± 7, [Na⁺] = 131.6 + 4.4, [K⁺] = 2.5 ± 0.7 and [CL⁻] = 110.6 ± 3.6. Freshwater acclimated fish gradually exposed to sea water (increase of 5 ppt h⁻¹) had higher plasma osmolalities than did the bay water acclimated fish. These young adult sturgeon are able to tolerate transfer from fresh water to sea water as well as gradual transfer from sea water to fresh water. Plasma electrolytes in transferred fish are regulated, but tend to differ from long term acclimated fish at the same salinities. There is a gradual increase in the upper salinity tolerance (abrupt transfer) of juvenile white sturgeon with weight: 5–10 ppt for 0.4–0.9 g fish, 10–15 ppt for 0.7–1.8 g fish, and 15 ppt for 4.9–50.0 g fish. The ability of juveniles to regulate plasma osmolality is limited. The young adult fish are able to tolerate higher salinities (35 ppt) than juvenile sturgeon but probably are also characterized by low activity of the necessary ion exchange mechanisms in the gills which permit rapid adjustment of blood electrolytes with graduate change in external salinity.     

CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> subgroups may be human small intestinal stem cells

The identification and separation of small intestinal epithelial stem cells are still on the preliminary stage. In this study, we planned to utilize immunohistochemistry, fluorescence-activated cell sorting (FACS) and RT-PCR to investigate the possibility of CD133 and CD44 as markers of human small intestinal epithelial stem cells. The expressions of CD133, CD44 and Lgr5 were studied by immunohistochemistry. Four subgroups of CD133⁺CD44⁺, CD133⁺CD44⁻, CD133⁻CD44⁺, CD133⁻CD44⁻ were sorted out through FACS and the expression level of Lgr5 gene was measured by RT-PCR and polyacrylamide gel electropheresis (PAGE) with sliver stained. Ten cases of samples were available for analyzing. By immunohistochemical staining, few cells with positive expressions of CD133, CD44 and Lgr5 were distributed in the bottom of crypts with the expression locations somewhat overlapped. The average percentage of CD133⁺CD44⁺ cells was 0.0580 ± 0.0403%, while the corresponding contents of CD133⁺CD44⁻ cells, CD133⁻CD44⁺ cells and CD133⁻CD44⁻ cells were 0.4000 ± 0.1225%, 0.7000 ± 0.2646% and 76.5600 ± 3.5529% respectively. Ten times of positive expressions of Lgr5 were detected in the CD133⁺CD44⁺ groups, while 9/10, 8/10 and 4/10 times for CD133⁺CD44⁻, CD133⁻CD44⁺ and CD133⁻CD44⁻ subgroups respectively. With the help of Quantityone 4.62 software, the densities of corresponding place to Lgr5 and reference gene were obtained. The density ratios of corresponding place to Lgr5 to reference gene were significant difference between subgroups (P < 0.001). By means of LSD method, the density ratios in CD133⁺CD44⁺ subgroups had statistical differences from the other subgroups (P < 0.05). We concluded CD133⁺CD44⁺ cells may be human small intestinal epithelial stem cells, which need further researches to confirm.     

Simultaneous nutrients and carbon removal during pretreated swine slurry degradation in a tubular biofilm photobioreactor

The biodegradation potential of an innovative enclosed tubular biofilm photobioreactor inoculated with a Chlorella sorokiniana strain and an acclimated activated sludge consortium was evaluated under continuous illumination and increasing pretreated (centrifuged) swine slurry loading rates. This photobioreactor configuration provided simultaneous and efficient carbon, nitrogen, and phosphorous treatment in a single-stage process at sustained nitrogen and phosphorous removals efficiencies ranging from 94% to 100% and 70–90%, respectively. Maximum total organic carbon (TOC), NH₄ ⁺, and PO₄ ³⁻ removal rates of 80 ± 5 g C m_r ⁻³ day⁻¹, 89 ± 5 g N m_r ⁻³ day⁻¹, and 13 ± 3 g P m_r ⁻³ day⁻¹, respectively, were recorded at the highest swine slurry loadings (TOC of 1,247 ± 62 mg L⁻¹, N–NH₄ ⁺ of 656 ± 37 mg L⁻¹, P–PO₄ ³⁺ of 117 ± 19 mg L⁻¹, and 7 days of hydraulic retention time). The unusual substrates diffusional pathways established within the phototrophic biofilm (photosynthetic O₂ and TOC/NH₄ ⁺ diffusing from opposite sides of the biofilm) allowed both the occurrence of a simultaneous denitrification/nitrification process at the highest swine slurry loading rate and the protection of microalgae from any potential inhibitory effect mediated by the combination of high pH and high NH₃ concentrations. In addition, this biofilm-based photobioreactor supported efficient biomass retention (>92% of the biomass generated during the pretreated swine slurry biodegradation).     

Chemiluminescent in vitro estimation of the inhibitory constants of antioxidants ascorbic and uric acids in Fenton’s reaction in urine

The goal of this research was to measure in vitro the inhibitory constants of the antioxidants ascorbic and uric acid in urine, with lucigenin enhanced chemiluminescence (CL) in Fenton’s system. Maximum CL emission is registered in urine containing H₂O₂ (5·10⁻⁴ M), Fe²⁺ (5·10⁻⁵ M), EDTA (5·10⁻⁵ M), and chemical enhancer lucigenin (10⁻⁴ M) at pH 5.5 and 36°C. Ascorbic acid exhibits up to 4-fold stronger antioxidant effect than uric acid. The constants of antioxidant inhibition in urine were measured at concentrations 10⁻³ and 10⁻⁴ M: for ascorbic acid, 5.92 ± 0.04 and 24.05 ± 1.82 μmol·sec⁻¹; for uric acid, 1.60 ± 0.02 and 21.45 ± 0.97 μmol·sec⁻¹, respectively. Three phases of CL kinetics of urine are well observed: spontaneous CL (0–10 sec), fast flash of CL (10–50 sec), and latent period (50–300 sec). The antioxidant efficiency of ascorbic and uric acids in the final stage of catabolic processes in the body is discussed. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 8, pp. 1062–1065.     

Chromate reduction by Rhodobacter sphaeroides

Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration. The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K _m of 15±1.3 μM CrO₄ ²⁻ and a V _max of 420±50 μmol min⁻¹ mg protein⁻¹ was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO₄ ²⁻ and 100±9.6 μmol CrO₄ ²⁻ min⁻¹ mg protein⁻¹ for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203. Received 05 January 2000/ Accepted in revised form 27 May 2000     

Isolation and cultivation of murine hematopoietic stem cells and expression of hFIX mediated by recombinant lentiviral vectors in vitro

Hematopoietic stem cells (HSCs) are an attractive target for gene therapy, especially for inherited blood diseases. Moreover, recombinant lentiviral vectors are considered to be prospective in HSCs gene therapy for the high efficiency of infection. In this study, murine mononuclear cells (MNCs) were isolated from bone marrow and cultured in suspension, and then Lin⁻CD117⁺ HSCs were isolated by immunomagnetic beads. During culturing, cells and colonies increased in HSCs supplied with cytokines while no change was observed in the control group without cytokines. FUXW recombinant lentiviral vectors were produced by calcium phosphate-mediated transient cotransfection infected MNCs from ICR and C57 mice. The hFIX expressions were 41.7 ± 4.2 ng / mL and 34.5 ± 6.6 ng/mL in supernatant on 7d. The hFIX expressions of HSCs infected by FUXW recombinant lentiviral vectors were 46.6 ± 5.7 ng/mL (with cytokines) and 33.3 ± 4.8 ng/mL (without cytokines) in supernatant on 7d. Results indicate that recombinant lentiviral vectors can infect murine MNCs and Lin⁻CD117⁺ HSCs efficiently, and expression of the transgene can be improved when supplied with cytokines. __________ Translated from Journal of Fudan University (Natural Science), 2005, 44(4): 503–506 [译自: 复旦学报(自然科学版), 2005, 44(4): 503–506]     

Development and application of real-time PCR for quantification of specific ammonia-oxidizing bacteria in activated sludge of sewage treatment systems

In this study, four real-time polymerase chain reaction (PCR) primer sets were developed for the 16S rRNA genes of specific ammonia-oxidizing bacteria (AOB) found in activated sludge of sewage treatment systems. The primer sets target two of several sequence types of the Nitrosomonas oligotropha cluster, members within the Nitrosomonas communis cluster, and all members of the Nitrosomonas europaea–Nitrosococcus mobilis cluster. The detection limit of each primer set was in the range of 3×10¹–6×10² genes reaction⁻¹. Reliable quantification of the target AOB DNA was obtained when the target AOB DNA comprised more than 0.1% of total AOB DNA in the sample. The application of the primer sets to samples taken from five sewage treatment systems showed that, in all systems, the majority of the AOB population was comprised of one sequence type of the N. oligotropha cluster (3.9±1.5×10⁹–1.7±0.5×10¹⁰ cell l⁻¹) and, in most systems, followed by members within the N. communis cluster (2.8±0.3×10⁹–1.0±0.1×10¹⁰ cell l⁻¹) or/and another sequence type of the N. oligotropha cluster (1.5±0.6×10⁸–5.5±0.5×10⁸ cell l⁻¹). N. europaea–N. mobilis cluster arose solely in small numbers (4.9±0.8×10⁸ cell l⁻¹) in one system. Real-time PCR-amplified products obtained from genomic DNA extracted from samples were verified using clone library, and it revealed that only the target AOB DNA were PCR amplified, without amplification of the nontarget sequences.     

Studies on water transport through the sweet cherry fruit surface: characterizing conductance of the cuticular membrane using pericarp segments

Water conductance of the cuticular membrane (CM) of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated by monitoring water loss from segments of the outer pericarp excised from the cheek of the fruit. Segments consisted of epidermis, hypodermis and several cell layers of the mesocarp. Segments were mounted in stainless-steel diffusion cells with the mesocarp surface in contact with water, while the outer cuticular surface was exposed to dry silica (22 ± 1 °C). Conductance was calculated by dividing the amount of water transpired per unit area and time by the difference in water vapour concentration across the segment. Conductance values had a log normal distribution with a median of 1.15 × 10⁻⁴ m s⁻¹ (n=357). Transpiration increased linearly with time. Conductance remained constant and was not affected by metabolic inhibitors (1 mM NaN₃ or 0.1 mM carbonylcyanide m-chlorophenylhydrazone) or thickness of segments (range 0.8–2.8 mm). Storing fruit (up to 42 d, 1 °C) used as a source of segments had no consistent effect on conductance. Conductance of the CM increased from cheek (1.16 ± 0.10 × 10⁻⁴ m s⁻¹) to ventral suture (1.32 ± 0.07 × 10⁻⁴ m s⁻¹) and to stylar end (2.53 ± 0.17 × 10⁻⁴ m s⁻¹). There was a positive relationship (r²=0.066**; n=108) between conductance and stomatal density. From this relationship the cuticular conductance of a hypothetical astomatous CM was estimated to be 0.97 ± 0.09 × 10⁻⁴ m s⁻¹. Removal of epicuticular wax by stripping with cellulose acetate or extracting epicuticular plus cuticular wax by dipping in CHCl₃/methanol increased conductance 3.6- and 48.6-fold, respectively. Water fluxes increased with increasing temperature (range 10–39 °C) and energies of activation, calculated for the temperature range from 10 to 30 °C, were 64.8 ± 5.8 and 22.2 ± 5.0 kJ mol⁻¹ for flux and vapour-concentration-based conductance, respectively. Received: 23 March 2000 / Accepted: 28 July 2000     

NMR Reinvestigation of the Caffeine–Chlorogenate Complex in Aqueous Solution and in Coffee Brews

Caffeine complexation by chlorogenic acid (3-caffeoylquinic acid, CAS Number [327-97-9]) in aqueous solution as well as caffeine–chlorogenate complex in freshly prepared coffee brews have been investigated by high-resolution ¹H-NMR. Caffeine and chlorogenic acid self-associations have also been studied and self-association constants have been determined resorting to both classical isodesmic model and a recently introduced method of data analysis able to provide also the critical aggregation concentration (cac). Furthermore, caffeine–chlorogenate association constant was measured. For the caffeine, the average value of the self-association constant determined by isodesmic model (K _i = 7.6 ± 0.5 M⁻¹) is in good agreement with the average value (K _a = 10 ± 1.8 M⁻¹) determined with the method which permits the determination of the cac (8.43 ± 0.05 mM). Chlorogenic acid shows a slight decreased tendency to aggregation with a lower average value of association constants (K _i = 2.8 ± 0.6 M⁻¹; K _a = 3.4 ± 0.6 M⁻¹) and a critical concentration equal to 24 ± 1 mM. The value of the association constant of the caffeine–chlorogenate complex (30 ± 4 M⁻¹) is compatible with previous studies and within the typical range of reported association constants for other caffeine–polyphenol complexes. Structural features of the complex have also been investigated, and the complex conformation has been rediscussed. Caffeine chemical shifts comparison (monomeric, complexed, coffee brews) clearly indicates a significant amount of caffeine is complexed in beverage real system, being chlorogenate ions the main complexing agents.     

Effects of attachment substrates on the growth and differentiation of LLC-PK1 cells

Summary The growth and differentiation of an established renal epithelial cell line, LLC-PK₁, on membrane bound mussel adhesive protein (MAP), collagen, and extracellular matrix (ECM) in serum-containing medium was studied. Cell attachment and growth on uncoated- vs. protein-coated cellulose nitrate and acetate membranes did not differ significantly, and confluence was achieved on all membranes. However, cells remained in a single monolayer only when plated on collagen or ECM. LLC-PK₁ monolayers grown on ECM-coated membranes displayed the highest transepitheliald-glucose transport (333 ± 22 ng·cm⁻²·min⁻¹) whereas cells plated on collagen-coated membranes displayed the lowest (94 ± 23 ng·cm⁻²·min⁻¹). Glucose flux values increased with age of the culture, reaching a plateau at 28 d postseeding. These results indicate that the underlying substratum and cell age can affect differentiation of renal epithelial cells in vitro.