Accumulation of sesquiterpenes and polysaccharides in cells of zedoary (Curcuma zedoaria Roscoe) cultured in a 10 L bioreactor

We developed a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe), using 100 g fresh weight inoculum in a batch culture. The maximum cell biomass of 68.46 g/L fresh weight was obtained after 14 days of culture in a 10 L bioreactor with a pitch-blade impeller maintained at an agitation speed of 150 rpm and an aeration rate of 2.5 L/min. The accumulation of sesquiterpenes and polysaccharide in zedoary cells from 2 to 18 days was measured by HPLC and a phenol-sulfuric acid assay, respectively. The total polysaccharide concentration increased between 2 to 10 days of culture and reached a maximum value of 6.55%. HPLC revealed several eluted peaks of sesquiterpenes, which increased in amplitude from days 2 to 10. Furthermore, our results indicated that biotransformation occurred in the cell suspension, transforming certain sesquiterpenes into other types during culture.     

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

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

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

DEVELOPMENTAL CHANGES IN THE COTYLEDONS OF LUPINUS LUTEUS L. DURING AND AFTER GERMINATION

Cotyledons of Lupinus luteus were sampled from 1 to 21 days after sowing and processed for light microscopy and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Length, width, and surface area of the cotyledons increased gradually until day 10. The thickness of the cotyledons increased from day 7 to day 12 and decreased thereafter. Morphometric analyses showed that the increase in length, width, and thickness of the cotyledon was due to cell expansion, and the decrease in thickness of the cotyledon was due to the decrease in the length of abaxial cells and in the total number of cells. Mesophyll development accompanied schizogenous and lysigenous air space formation. There were two structurally distinct types of protein bodies. Protein bodies in five to six layers of cells on the abaxial side did not contain globoids, while globoids were prominent in protein bodies in the center and adaxial side. Storage protein mobilization occurred first in the abaxial side of the cotyledon and proceeded toward the adaxial side. SDS-PAGE analysis showed that proteins ranged from 97 to 14 kD. High molecular weight α- and ß-conglutinins were more abundant in the abaxial region, whereas γ-conglutinin occurred in both abaxial and adaxial regions. In addition, there were five minor bands between 97 and 43 kD unique to abaxial region and five minor bands between 43 and 14 kD unique to adaxial region in the nonreduced protein profiles. The α- and ß-conglutinins began to decrease after imbibition and disappeared by day 7 after sowing. At this stage the subunits of ribulose-l,5-bisphosphate carboxylase/oxygenase and four new minor bands appeared.     

Altering Developmental Trajectories in Mice by Restricted Index Selection

A restricted index selection experiment on mice was carried out for 14 generations on rate of early postnatal development (growth rate from birth to 10 days of age) vs. rate of development much later in ontogeny (growth rate from 28 to 56 days of age). Early rate of development (E) approximates hyperplasia (changes in cell number) and later rate (L) reflects hypertropy (changes in cell size). The selection criteria were as follows: E+L0 was selected to increase early body weight gain while holding late body weight gain constant; E-L0 was selected to decrease early body gain while holding late gain constant; E0L+ was selected to increase late gain holding early gain constant; and E0L- was selected to decrease late gain holding early gain constant. After 14 generations of selection, significant divergence among lines has occurred and the changes in the growth trajectories are very close to expectation. The genetic and developmental bases of complex traits are discussed as well as the concept of developmental homoplasy.     

Cell Wall Metabolism in Developing Strawberry Fruits

Cell wall metabolism was studied in strawberry receptacles (Fragariaananassa, Duchesne) of known age in relation to petal fall (PF).Polysaccharide and protein composition, incorporation of [¹⁴C]glucoseand [¹⁴C]proline by excised tissue, and the fate of ¹⁴CO₂ fixedby young, attached fruits were followed in relation to celldivision, cell expansion, fine structure, and ethylene synthesis. Cell division continued for about 7 d after PF although vacuolationof cells was already beginning at PF and the subsequent cellexpansion was logarithmic. There was an associated logarithmicincrease in sugar content per cell and a decreasing rate ofethylene production per unit fresh weight. During cell expansion radioactivity from [¹⁴C]glucose was incorporatedinto fractions identified as starch and soluble polyuronideand into glucose and galactose residues in the cell wall. Radioactivityfrom [¹⁴C]proline was also incorporated into the cell wall,but only 10 per cent of this activity was found in hydroxyproline.Correspondingly wall protein contained a low proportion of hydroxyprolineresidues. The proportion of radioactivity from ¹⁴CO₂ fixed byfruitlets remained constant in most sugar residues in the cellwall. The proportion of radioactivity in galactose fell, indicatingturnover of these residues. Between 21 and 28 d after PF receptacles became red and softenedbut there was no change in the rate of ethylene production.Cell expansion continued for at least 28 d. Tubular proliferationof the tonoplast and hydration of middle lamella and wall matrixmaterial had begun 7–14 d after PF but became extremeduring ripening. Associated with the hydration of the wall,over 70 per cent of the polyuronide in the wall became freelysoluble, and arabinose and galactose residues lost from thewall appeared in soluble fractions. There was no increase intotal polysaccharide during ripening and incorporation of [¹⁴C]glucoseinto polysaccharides ceased, although protein increased andincorporation of [¹⁴C]proline into wall protein continued.     

Effect of the duration of infusion of urogastrone* on intestinal regeneration in rabbits

Abstract Urogastrone (UG) increases the rate of mucosal regeneration on patched intestinal defects. Our aim was to determine the effect of the duration of UG administration on regeneration of serosa patched ileal defects in rabbits. Group I (n= 18) were controls. Group II (n= 15), Group III (n= 10) and Group IV (n= 5) received UG 1.5 μ/kg/h subcutaneously for 7 days, 14 days or 21 days respectively. Animals were sacrificed at 7 day intervals up to 21 days after patching. Neomucosal growth was significantly greater in the animals receiving UG and was greatest in Group IV. Group II and III animals had less contraction of the patched defect and greater neomucosal surface area than Group I animals at each interval but had a lesser effect than animals receiving UG continuously. Crypt cell production rate was significantly greater in UG treated animals at 7 and 14 days but fell to control levels at 21 days. Prolonging the duration of UG infusion increased the quantity of neomucosa produced by intestinal regeneration. However, UG stimulation of mucosal cell migration and proliferation occurred transiently within 14 days after patching.     

Effect of Growth Rate and Starvation-Survival on the Viability and Stability of a Psychrophilic Marine Bacterium

Cell populations of the marine bacterium ANT-300, from either batch or continuous culture with dilution rates ranging from D = 0.015 h⁻¹ to D = 0.200 h⁻¹, were monitored for viability, direct counts, and optical density for 98 days under starvation conditions. Three stages of starvation survival were observed for each of the cell populations. Although direct counts remained at 2 × 10⁷ to 3 × 10⁷ cells ml⁻¹ throughout the starvation period, large fluctuations occurred in cell viability during stage 1 (0 to 14 days) of starvation survival. Stage 2 (14 to 70 days) involved an overall decrease in viability for each of the cell populations; the rate of viability loss was dependent upon the growth rate. Cell viability stabilized at approximately 0.3% of the direct count in stage 3 (70 to 98 days). Long-term starvation corresponded to the prolongation of stage 3 starvation survival. Cell volumes for each of the cell populations decreased with the length of the starvation period. However, the cell volume of starved cells was also dependent more on growth rate than on the length of the time starved. We hypothesize that the cell population with the slowest growth rate is most closely representative of cells found in the oligotrophic marine environment.     

Monolayer expansion induces an oxidative metabolism and ROS in chondrocytes

This study tests the hypothesis that articular chondrocytes shift from a characteristically glycolytic to an oxidative energy metabolism during population expansion in monolayer. Bovine articular chondrocytes were cultured in monolayer under standard incubator conditions for up to 14 days. Cellular proliferation, oxygen consumption, lactate production, protein content, ROS generation and mitochondrial morphology were examined. Lactate release increased ∼5-fold within 1 week, but this was limited to ∼2-fold increase when normalized to cellular protein content. By contrast, per cell oxidative phosphorylation increased 98-fold in 1 week. The increase in oxidative phosphorylation was evident within 24 h, preceding cell proliferation and was associated with augmented reactive oxygen species generation. The autologous chondrocyte implantation procedure requires 14-21 days for population expansion. The alterations in metabolic phenotype we report within 7 days in vitro are thus pertinent to autologous chondrocyte implantation with significant implications for the chondrocyte functionality.     

Macrophage populations and cardiac sympathetic denervation during L-NAME-induced hypertension in rats

The rat model of hypertension induced by prolonged treatment with Nomega-nitro-L-arginine methyl ester (L-NAME) has been extensively used. However, the effects on cardiac autonomic innervation are unknown. Here, the cardiac sympathetic innervation is analyzed in parallel with myocardial lesions and leukocyte infiltration during L-NAME (40 mg/Kg body weight/day, orally) treatment. The occurrence of cardiomyocyte hypertrophy, a controversial matter, is also addressed. Degenerating cardiomyocytes and focal inflammation occurred one day after treatment. Inflammatory lesions became gradually more frequent until day 7. At day 14 fibroblast-like cells were outstanding. Interstitial and perivascular connective tissue increased from day 28 on. In the left ventricle, cardiomyocyte hypertrophy occurred only around the damaged area during the first 14 days. After 28 days, it became more widespread. In the right ventricle, the hypertrophic cardiomyocytes were restricted to damaged areas. Significant reduction of the noradrenergic nerve terminals occurred from day 3 to 28. The area occupied by ED1+ (hematogenous) macrophages increased until day 7, and dropped to control levels by day 10. ED2+ (resident) macrophages increased from day 3 to 7 and remained higher than control values up to day 77. Animals receiving both L- NAME and aminoguanidine (AG), an inducible nitric oxide synthase (iNOS) inhibitor (65 mg/Kg body weight/day, orally), showed significant decrease in the nitrite serum levels, sympathetic denervation and macrophage infiltration at day 7. No denervation was detectable at day 14 of double treatment, using subcutaneous AG. Our findings favor a role for ED1+ macrophages and iNOS in the hypertension-induced denervation process.     

Biosynthesis of glycosaminoglycans in the developing retina

The glycosaminoglycans of neural retinas from 5-, 7-, 10-, and 14-day chick embryos were labeled in culture with [³H]glucosamine and ³⁵SO₄, extracted, and isolated by gel filtration. The incorporation of label per retina into glycosaminoglycans increased with embryonic age, but that per cell and per unit weight of uronic acid decreased. Specific enzyme methods coupled with gel filtration and paper chromatography demonstrated that [³H]glucosamine incorporation into chondroitin sulfate increased between 5 and 14 days from 7 to 34% of the total incorporation into glycosaminoglycans. During this period, incorporation into chondroitin-4-sulfate increased relative to that into chondroitin-6-sulfate. Between 5 and 10 days, incorporation into heparan sulfate showed a relative decline from 89 to 61%. Incorporation into hyaluronic acid always represented less than 2% of the total. A twofold greater increase in galactosamine concentration than in glucosamine concentration in the glycosaminoglycan fraction between 7 and 14 days supports the conclusion that chondroitin sulfate was the most rapidly accumulating glycosaminoglycan. ECTEOLA-cellulose chromatography revealed a heterogeneity in the size and/or net charge of chondroitin sulfate and heparan sulfate. We conclude that incorporation of exogenous precursors into glycosaminoglycans in the chick retina decreases relative to cell number as differentiation progresses from a period of high mitotic activity to one of tissue specialization, and that it is accompanied by a net accumulation of glycosaminoglycan and a change in the pattern of its synthesis.     

Biphasic changes in heart performance with food restriction in rats.

To examine effects of food restriction resembling very-low-calorie dieting on heart performance, normal rats were fed 25% of ad libitum food intake for 14 days. Although heart weight decreased (P < 0.05) after 5 days, left ventricular systolic pressure as well as rates of pressure development and fall were increased (P < 0.05) at 7 days and decreased (P < 0.05) after 14 days. Systolic and diastolic blood pressures were also increased from 5 to 7 days and decreased after 14 days. The increased hemodynamic performance of heart was associated with a raised plasma norepinephrine concentration, which peaked at day 7 of food restriction; epinephrine concentration was increased (P < 0.05) also at day 7. An increased catecholamine synthesis was indicated by the raised (P < 0.05) plasma dopamine beta-hydroxylase activity at 3 days, but this was decreased (P < 0. 05) at 14 days. The concentration of dopamine in the heart was increased (P < 0.05) at 2-14 days, of norepinephrine at 7-14 days, and of epinephrine at 10 and 14 days. Food restriction thus appears initially to be associated with an enhanced catecholamine influence on the heart and is followed by a depressed cardiac performance.     

The effect of oxygen deficiency on uptake and distribution of nutrients in maize plants

Young maize (Zea mays L.) plants, 7 days after germination were exposed to nutrient solutions which were either aerated or not aerated for 14 days. Nutrients were supplied as 50% strength Hoagland’s solution or, in the case of the four ‘low nutrient’ treatments, N, P, K or Ca were supplied at the equivalent of 10% strength Hoagland’s solution. Shoot fresh weight was decreased by 25% due to lack of aeration; O₂ deficiency also impaired leaf elongation but not dry weights, suggesting that lack of O₂ in the roots impaired cell expansion in shoots more than dry weight accumulation. The distribution of N, P, K and Ca within shoots was consistent with their relative mobilities in the phloem; at least 7% of Ca in plants after 14 days of treatments was found in the oldest leaf whereas N, P and K were rapidly remobilised to younger tissues. Between 33 and 49% of the total N, P and K in the shoot was found in the 40 mm of tissue at the base of the growing leaves in plants grown for 14 days at low nutrient concentrations. Concentrations (dry weight basis) of phloem-mobile nutrients were also greatest in the growing zones of the leaves, especially in the case of N and P. Calcium, on the other hand, was found in relatively low concentrations in the youngest tissue and as with the other nutrients, concentrations declined due to low external supply, non-aeration or a combination of both. In spite of the failure of Ca to move from old to young leaves, the effect of the deficiencies of N, P and K was probably as severe as that of Ca in the youngest tissues of treated plants. Calcium uptake by the whole shoot appeared to be slightly less sensitive to O₂ deficits than that of N, P and K. This compensated for the failure of Ca to move to growing tissues during periods of low external Ca supply.     

On the role of 17 alpha-estradiol and 17 beta-estradiol in the proliferation of MCF7 and T47D-A11 human breast tumor cells

A comparative study of the proliferative effect of 17 beta-estradiol and 17 alpha-estradiol on human estrogen-sensitive cell lines was performed. When using charcoal-dextran stripped human female sera-supplemented media the administration of the hormones, 17 alpha-estradiol at 3 X 10(-10)M, and 17 beta-estradiol at 3 X 10(-11)M, resulted in a ten-fold increase in cell yield when compared with non-estrogen supplemented controls after cells were grown for periods between 10 to 14 days. No significant metabolization of 17 alpha-estradiol into 17 beta-estradiol occurred as measured by the E2 levels in the supernatants of the cell culture flasks. Increased concentrations of 17 beta-estradiol and 17 alpha-estradiol added to the media bathing C7MCF7-173 cells resulted in a triggering of a partially successful shut-off effect; this phenomenon was not observed with T47D-All cells. These results are compatible with predictions stemming from the indirect and direct negative working hypothesis for the regulation of cell proliferation.     

Postnatal changes in the DNA content of mouse cerebral hemispheres.

Changes in the wet weight of the cerebral hemispheres and in their DNA content and concentration were studied in CBA mice (non-SPF, Velaz, Prague) aged from 1 to 270 days. It was found that hemisphere wet weight rose by 350% between the 1st and 14th day and by a further 11% between the 14th and 180th day. In the next three months it remained stable. The total DNA content rose by 30% between the 1st and 2nd day and by 45% between the 1st and 10th day; changes between the 10th and 180th day were non-significant, but a decrease of 16% occurred by the 270th day. Between the 1st and 2nd day the DNA concentration did not alter, or rose non-significantly (+20%). Towards the end of the 2nd postnatal week it fell exponentially (-75%). Changes in the DNA content and concentration indicate that the rate of cell proliferation in mouse cerebral hemispheres is highest on the first two days after birth, while the general chemical composition of the hemisphere develops fastest between the 2nd and 14th day. The constancy of the DNA content between the 10th and 180th day implies that cell division in the hemispheres of adolescent and adult mice primarily reflects renewal of the non-neuronal cell population.     

Plant Regeneration from Protoplasts of Actinidia chinensis Planch.

Protoplasts isolated from cotyledon callus line of A14N7 of Actinidia Chinensis Planch. were cultured in the improved NN-69 medium. First division of regenerated cells occurred during 7–10 days of culture, and percentage of the cell division was about 10% at day 20. The best result of protoplast culture was achieved when protoplasts were cukured in liquid medium at a density of 5× 104/ml, About 4 months, procoplast-derived calli were transferred stepwisely onto differentiation media where they developed into green compact calli, from which the perfect plants were regenerated.     

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

R-Spondin1 enhances wnt signaling and decreases weight loss in short bowel syndrome zebrafish

BackgroundR-spondins, including R-spondin 1 (RSPO1), are a family of Wnt ligands that help to activate the canonical Wnt/β-catenin pathway, which is critical for intestinal epithelial cell proliferation and maintenance of intestinal stem cells. This proliferation underpins the epithelial expansion, or intestinal adaptation (IA), that occurs following massive bowel resection and short bowel syndrome (SBS). The purpose of this study was to identify if recombinant human RSPO1 (rhRSPO1) could be serially administered to SBS zebrafish to enhance cellular proliferation and IA.MethodsAdult male zebrafish were assigned to four groups: sham + PBS, SBS + PBS, sham + rhRSPO1, and SBS + rhRSPO1. Sham fish had a laparotomy alone. SBS fish had a laparotomy with distal intestinal ligation and creation of a proximal stoma. Fish were weighed at initial surgery and then weekly. rhRSPO1 was administered post-operatively following either a one- or two-week dosing schedule with either 3 or 5 intraperitoneal injections, respectively. Fish were harvested at 7 or 14 days with intestinal segments collected for analysis.ResultsRepeated intraperitoneal injection of rhRSPO1 was feasible and well tolerated. At 7 days, intestinal epithelial proliferation was increased by rhRSPO1. At 14 days, SBS + rhRSPO1 fish lost significantly less weight than SBS + PBS fish. Measurements of intestinal surface area were not increased by rhRSPO1 administration but immunofluorescent staining for β-catenin and gene expression for cyclin D1 was increased.ConclusionsIntraperitoneal injection of rhRSPO1 decreased weight loss in SBS zebrafish with increased β-catenin + cells and cyclin D1 expression at 14 days, indicating improved weight maintenance might result from increased activation of the canonical Wnt pathway.     

Serotonin as a growth factor for chick embryo brain

It has been suggested that biogenic amines, in addition to their role as neurotransmitters, may also act as growth factors in the embryo. In the present work the concentrations of serotonin (5-HT), norepinephrine (NE) and dopamine (DA) were first determined in cerebral hemispheres of chick embryo at 10, 12 and 14 days of continuous incubation at 37.5°C (controls). When the incubation on days 7–10 was at 40°C (experimentals), a procedure known to increase brain weight, brain protein content and brain cell number, the concentration of 5-HT in cerebral hemispheres at day 10 (end of neuron proliferation) was significantly increased; this increase persisted at days 12 and 14 but ceased to be significant. No such increases were observed in the concentrations of NE and DA in experimentals at either day 10, 12 or 14. When 5-HT was injected into albumen of eggs at day 7 (37.5°C), cerebral weights, optic lobe weights and cerebral concentrations of 5-HT at day 10 were significantly increased over non-injected controls. Elevated temperature of incubation (40°C) further increased cerebral weight and 5-HT concentration. Cerebral protein contents and the ratios of cerebral protein/ cerebral DNA at day 10 were also significantly increased but cerebral DNA and body weights were unchanged. The optimal doses have been determined. It is concluded that 5-HT may be a growth promoting or regulating factor for embyronal brain.