Hydroxyapatite-pulp composite fiber sheet bed: A new material for the immobilization of CHO-K1 cells

A new immobilization material for cell culture, ahydroxyapatite-pulp composite fiber (HAPC) sheet bed, was usedto grow CHO-K1 cells. The sheet bed for cell culture wasprepared from HAPC fiber by paper-making techniques. Scanning electron microscopic analysis revealed that the HAPCsheet bed had a structure consisting of piled fibers with spaces 10–200 m in diameter and a pore surface area of 0.32 m² g^-1. Using a 25 × 25 mm² squareHAPC sheet bed 0.41 mm in thickness (85 g m^-2 basis weight) for cell culture, CHO-K1 cells grew to a cell densityof 3.7 × 10⁷ cells cm^-3 in a 60 mm plastic dish over a 6-day culture period. High-density culture of CHO-K1 cells was successfully performed using the HAPC sheet bed in a 500 ml spinner flask over a 21-day culture period. The HAPC sheet bed, wound around the stirrer paddle, was rotated in the spinner flask in order to supply nutrientsand remove waste products efficiently. The HAPC sheet bedhas a large surface area to support cell growth and there islarge diffusion space inside of the bed. This newautoclavable substrate for anchorage-dependent cells can be easily scaled-up.     

A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes

Summary To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2×2×2 mm, mean pore size: 100, 250 or 500 m) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6–11.3×10⁷ hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2×10⁷ cells/cm³ PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes. Correspondence to: N. Ohshima     

Long term culture and caffeine production of immobilized coffee (Coffea arabica) L. cells in polyurethane foam

Coffee (Coffea arabica L.) cells could be immobilized in polyurethane foam and subcultured repeatedly for a long time. Four phases were observed for cell growth and caffeine production, I; immobilization, II; growth, III; caffeine production, IV; regrowth. Their periods were influenced by the number of foam particles. Especially in the phase III, the immobilized cells produced a relatively large amount of caffeine in the subculture numbers 5–8 (34 cubes) when the fresh weight of the immobilized cells decreased despite culture in growth medium (DK medium). Caffeine production appeared to have a negative correlation with the growth of the immobilized cells throughout the subcultures.This paper is Part 71 in the series of Studies on Plant Tissue Cultures. For Part 70, see Orihara Y, Furuya T. submitted for publication.     

Immobilisation of anchorage-independent animal cells using reticulated polyvinyl formal resin biomass support particles

Summary Immobilisation of anchorage-independent animal cells using Biomas Support Particles (BSPs) was investigated. Mouse myeloma MPC-11 cells were physically entrapped in three-dimensional reticulated polyvinyl formal (PVF) resin PSPs (3×3×3 mm) with matrices of relatively small pores (30–100 m) by filtering medium containing cells or incubating in a shake flask for inoculation. Physically entrapped cells became immobilised in the BSPs by forming aggregates within the matrices of the reticulated PVF resin, and cell density in the BSPs reached at least 10⁷ cells/cm³ BSP. Immobilised cells in the BSPs were successfully cultivated in static and/or shake-flask cultures with regular replacement of medium for a long period.     

Fluorescence microscopy and radiolabeling of C3 and C4 chloroplasts using diisothiocyanatostilbene disulfonic acid as a marker for the phosphate translocator

The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C₃ plant) and maize (Zea mays L.) (C₄ plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [³H₂]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, M_r, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [³H₂]DIDS. However, when broken chloroplasts were incubated with [³H₂]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [³H₂]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C₃ and C₄ chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB Coomassie brilliant blue - DIC differential interference contrast optics - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - [³H₂]DIDS 1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane - kDa kilodalton - M_r relative molecular mass - PGA 3-phosphoglycerate - Pitranslocator phosphate translocator - SDS sodium dodecyl sulfate     

Characteristics of immobilized Rhizopus oryzae in polyurethane foam cubes

Summary Rhizopus oryzae was immobilized in polyurethane foam cubes. The effects of the cube size on cell immobilization, cell growth and L(+)-lactic acid production were studied. By the natural attachment method, R. oryzae could be easily immobilized in the polyurethane foam cubes larger than 2.5 × 5 × 5 mm³. The use of small cubes for R. oryzae immobilization was very effective to increase the productivity of L(+)-lactic acid by the immobilized cells. Although it was difficult for smaller cubes to be completely full of the mycelia, increasing the inoculum size in immobilizations was effective to increase the immobilization ratio (a ratio of the number of the cubes containing cells to the total number of cubes).     

Limnology of Crater Lakes in Los Tuxtlas,Mexico

Investigations on the abundance, biomass and position of heterotrophic flagellates (HF) in the benthic microbial food web of a melt water stream on King George Island, Antarctic Peninsula, were undertaken during the Antarctic summer from 23rd December 1997 until 13th March 1998. Abundance and biomass of potential HF resources (picophotoautotrophic and non-photoautotrophic bacteria) as well as potential predators on HF (ciliates and meiofauna) were also investigated. HF abundance ranged from approximately 9 × 10³ to 81 × 10³ cells cm^–3, values which fall into the same range as those found in lower latitudes. Numerically important benthic HF were euglenids, kinetoplastids, thaumatomastigids and especially chrysomonads. Most species identified have been shown to have a worldwide distribution. Abundance of the benthic ciliates ranged from 27 to 950 cells cm^–3. Mean bacterial abundance was 1.9 × 10⁷ and 5.2 × 10⁸ cells cm^–3 for picophotoautotrophic and non-photoautotrophic benthos, respectively. The well-developed microbial community was able to support the large number of nematods, gastotrichs, tardigrads and rotifers with abundances reaching more than 1000 individuals cm^–3. The largest portion of heterotrophic biomass was formed by the meiofauna with a mean of 63 g C cm^–3, followed by that of the heterotrophic bacteria with 4.80 g C cm^–3. Picophotoautotrophic bacteria contributed a mean of 1.37 g C cm^–3. HF and ciliates mean biomass was 0.61 and 1.99 g C cm^–3, respectively, with the HF biomass comprising between <10 and 70% of the total protozoan biomass. The data obtained in this study identify the melt water stream as a hot-spot of heterotrophic microbial and meiofaunal activity during the austral summer. The HF in the melt water stream formed a diverse group in terms of taxa and potential feeding types. Chrysomonads, kinetoplastids, euglenids and thaumatomastigida were the most abundant taxa. A classification into feeding types identified an average of 34% of the total HF as bacterivorous while all others were able to utilise other, larger organisms as resources. Potential trophic interactions between HF and bacteria and higher trophic levels are discussed.     

Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds

Mean pore size is an essential aspect of scaffolds for tissue-engineering. If pores are too small cells cannot migrate in towards the center of the construct limiting the diffusion of nutrients and removal of waste products. Conversely, if pores are too large there is a decrease in specific surface area available limiting cell attachment. However the relationship between scaffold pore size and cell activity is poorly understood and as a result there are conflicting reports within the literature on the optimal pore size required for successful tissue-engineering. Previous studies in bone tissue-engineering have indicated a range of mean pore sizes (96–150 µm) to facilitate optimal attachment. Other studies have shown a need for large pores (300–800 µm) for successful bone growth in scaffolds. These conflicting results indicate that a balance must be established between obtaining optimal cell attachment and facilitating bone growth. In this commentary we discuss our recent investigations into the effect of mean pore size in collagen-glycosaminoglycan (CG) scaffolds with pore sizes ranging from 85–325 µm and how it has provided an insight into the divergence within the literature.Key words: bone tissue engineering, cell adhesion, collagen, extracellular matrix, pore size, scaffoldThe goal of tissue engineering is to develop cell, construct and living system technologies to restore the structure and functional mechanical properties of damaged or degenerated tissue. While the field of tissue engineering may be relatively new, the idea of replacing tissue with another goes as far back as the 16^th century when an Italian, Gasparo Tagliacozzi (1546–99), Professor of Surgery and Anatomy at the Bologna University, described a nose replacement that he had constructed from a forearm flap in his work “De Custorum Chirurigia per Insitionem” (The Surgery of Defects by Implantation) which was published in 1597. In modern times, the techniques of transplanting tissue from one site to another in the same patient (an autograft) or from one individual to another (transplant or allograft) have been revolutionary and lifesaving. However major problems exist with both techniques. Harvesting autografts is expensive, painful, constrained by anatomical limitations and associated with donor-site morbidity due to infection and hemorrhage. Transplants have serious constraints. The major problem is accessing enough tissue and organs for all of the patients who require them. Transplants are strongly associated with rejection by the patient''s immune system and they are also limited by the potential risks of introducing infection or disease.Tissue engineering was born from the belief that primary cells could be isolated from a patient, expanded in vitro and seeded onto a substrate that could be grafted back into the patient.¹ It provides a biological alternative to transplantations and prosthesis. One of the first scaffolds pioneered for tissue regeneration was synthesized as a graft co-polymer of type I collagen and chondroitin 6-sulphate, a glycosaminoglycan. The development of these scaffolds, which are capable of supporting tissue synthesis when seeded with cells, marks the beginning of the field of tissue engineering.^2,3 Since this early work, there have been rapid advances in bone tissue engineering with the development of porous, biocompatible, three-dimensional scaffolds. Regardless of the application, the scaffold should be biocompatible and imitate both the physical and biological function of the native extracellular matrix (ECM), as the ECM provides a substrate with specific ligands for cell adhesion as well as physical support for cells.⁴ When designing scaffolds for any tissue engineering application, a major consideration is the mean pore size. Scaffolds must be permeable with interconnecting pores to facilitate cell growth, migration and nutrient flow. A previous study demonstrated that permeability increases with increasing pore size due to a reduction in specific surface area.⁵ If pores are too small, cell migration is limited, resulting in the formation of a cellular capsule around the edges of the scaffold. This in turn can limit the distribution of nutrients and removal of waste products resulting in necrotic regions within the construct. Conversely if pores are too large there is a decrease in specific surface area.³ It has been proposed that a reduction in specific surface area reduces the ligand density available for cells to bind to.⁶ Cellular activity is influenced by specific integrin-ligand interactions between cells and surrounding ECM. Initial cell adhesion mediates all subsequent events such as proliferation, migration and differentiation within the scaffold. As a result the mean pore size within a scaffold affects cell adhesion and ensuing proliferation, migration and infiltration. Therefore maintaining a balance between the optimal pore size for cell migration and specific surface area for cell attachment is essential.^4,7In our laboratory we use a composite scaffold fabricated from collagen and a glycosaminoglycan (GAG) for bone tissue engineering applications produced by a lyophilisation (freeze-drying) fabrication process. The first generation of this collagen-GAG (CG) scaffold was originally developed for skin regeneration but has since been applied to a number of other tissue engineering applications, due to its high biological activity and resultant ability to promote cell growth and tissue development.^2,8–12 Originally CG scaffolds were fabricated using a rapid uncontrolled quench process during lyophilisation which resulted in heterogeneous porous scaffolds with a large variation of pore size within certain areas of the scaffold.² When these scaffolds were used in previous studies they were visually examined so that the areas of variation could be avoided resulting in subjective selection of scaffold samples for analysis.⁸ However, an improved lyophilisation technique was later developed which incorporated a constant cooling rate which controlled the formation and growth of ice-crystals thus resulting in CG scaffolds with homogenous pore structures.¹³ The traditional final temperature of freezing used to produce these scaffolds is −40°C; however, further modifications to the lyophilisation process demonstrated that by changing the final temperature of freezing, it is possible to tailor the mean pore size in the scaffolds. This study showed that by varying the temperature of freezing from −40 to −10°C it was possible to produce homogenous CG scaffolds with mean pore sizes ranging from 96–151 µm.⁶A cellular solid is one made up of an interconnecting porous network and cellular solids modeling techniques can be used to describe both mechanical and microstructural (i.e., specific surface area) properties of scaffolds. A cellular solids model utilizing a tetrakaidecahedral unit cell (a 14-sided polyhedron that packs to fill space) was used to determine the effect of mean pore size on specific surface area. Specific surface area can be related to the relative density of a scaffold and using a tetrakaidecahedral unit cell it was possible to model the geometry of the CG scaffolds.^5,6,14 As a result the specific surface area (SA) per unit volume (V) available for cell adhesion in each of the scaffolds with different mean pore sizes (d) was estimated as: SA/V = 0.718/d(1)This relationship demonstrates that the specific surface area is inversely proportional to the mean pore size. The authors then carried out a simple experiment and seeded the scaffold range with osteoblasts and monitored initial cell adhesion up to 48 h post-seeding. Cell adhesion is the binding of cells to their extracellular environment via specific ligand-integrin interactions. The results demonstrated that cell adhesion decreased with increasing pore size and that the highest levels of cell attachment were found on the scaffolds with the smallest pore size (96 µm). The rationale for this result, as suggested by the authors, was the effect of specific surface area on cell adhesion due to the scaffolds with larger pores having less available specific surface area and thus a lower ligand density for initial cell attachment.^5,6The results of this study conflicted with other studies within the literature which demonstrate a need for larger pores. The relationship between scaffold pore size and cell activity is not fully understood and as a result, over the years there have been conflicting reports on the optimal pore size required for bone tissue engineering. Pores ranging from 20–1,500 µm have been used in bone tissue engineering applications.^15–18 Initial studies demonstrated that the minimum pore size for significant bone growth is 75–100 µm with an optimal range of 100–135 µm.^15,19 Since this early work it has been reported that pores greater than ∼300 µm are essential for vascularisation of constructs and bone ingrowth, while pores smaller than ∼300 µm can encourage osteochondral ossification.^20–22In a very recent study in our laboratory, which utilized improved technical capability of our freeze-drying system and introduced a novel annealing step during lyophilisation, we have been able to further expand the range of mean pore sizes produced in the CG scaffolds from 96–151 µm up to 85–325 µm.²³ We then investigated the effect of this new expanded range of scaffolds on initial cell attachment followed by migration and proliferation by monitoring cellular activity up to 7 days post-seeding (as opposed to 48 h in the earlier study⁶) to see whether the pattern of specific surface area affecting initial cell adhesion as seen in the previous studies would continue as cells proliferated.²⁴The results provide a possible insight into why there are conflicting reports in the literature on the optimal scaffold pore size for bone tissue engineering. A non-linear effect of pore size was seen on cell proliferation over the 7 day incubation period. Scaffolds with the largest pore size of 325 µm facilitated higher cell number at all time points in comparison to the other scaffold types. However, within the lower range of pore sizes there was a small peak in cell number at 24 h and 48 h post-seeding in scaffolds with a mean pore size of 120 µm. This peak disappeared by day 7 (Fig. 1). This peak is consistent with that seen in the earlier study⁶ and can therefore be explained by the effect of pore surface area on cell attachment. Collagen, a natural component of bone ECM, contains binding sites (ligands) that are recognized by specific cell surface receptors (integrins), the main collagen integrins being α₁β₁ and α₂β₁. Based on the interactions between integrins and their corresponding ligands, cells can detect subtle changes in ECM that can influence cell attachment and consequently determine cell proliferation, speed and migration. Our results reflected this within the smaller pore range (85–190 µm) when cell number was presented as a percentage of the cells seeded onto the scaffolds,²⁴ indicating that high specific surface area in scaffolds is important for optimal cell attachment. However, when this range of pore sizes was expanded (85–325 µm) the linear relationship between mean pore size and specific surface area was no longer applicable (Fig. 1) and scaffolds with the largest pores showed the highest cell numbers even though the surface area is lower than that for the other scaffold variants. We propose that the effect of specific surface area is overcome in larger pores by the improved potential for cell migration and proliferation as was seen histologically in scaffolds with 325 µm.Open in a separate windowFigure 1Effect of mean pore size on cell number at each time point. Cell number increases to a small peak 24 h post seeding in scaffolds with a pore size of 120 µm. This peak declines at later time points. Cell number significantly peaks in scaffolds with a mean pore size of 325 µm. *p < 0.001 (reviewed in ref. ²⁴).When seeding three-dimensional scaffolds it is desirable that the cells infiltrate and colonize the scaffold laying down their own ECM. The CG scaffolds are highly porous (∼99%)⁵ and it has previously been shown that cell migration behavior decreases with increasing pore size.²⁶ However, similarly to other studies,⁶ these results were based on limited range of mean pore sizes incubated for less than 48 h. In this study, migration of cells was assessed histologically after 7 days incubation. Cells were observed lining the pores in all scaffolds. However, cell aggregations were seen along the edges of the scaffolds with smaller pore sizes of 85 µm–120 µm limiting the number of cells infiltrating the scaffold (Fig. 2A). Cell aggregations form a “skin” around the outer surface of the scaffold which restricts the diffusion of nutrients and removal of waste from the cells colonizing the center of the scaffold. As the mean pore size increased, cells migrated further away from the edges and in towards the center of the scaffold until cells were seen colonizing the center of the scaffolds with the largest mean pore size of 325 µm (Fig. 2B). An increase in cell number was seen in 120 µm pore size, but the aggregations seen on the surface of these scaffolds compound the hypothesis that this peak was related to initial cell adhesion and the advantages of this pore size were lost with subsequent cell proliferation and migration.Open in a separate windowFigure 2Effect of mean pore size on cell infiltration and distribution CG scaffolds after 7 days. Scaffolds were stained with H&E: (A) 85 µm pore size at x40 magnification, (B) 325 µm pore size at ×40 magnification. Collagen scaffold is stained pink and cell nuclei a deep purple. The arrow indicates cell aggregations along the edges of the scaffold. Aggregations disappeared and cell migration increased with increasing pore size (reviewed in ref. ²⁴).The study²⁴ had a number of limitations. It was not possible to determine the upper pore size limit for cell activity within a CG scaffold. If the pores become too large the mechanical properties of the scaffold will be compromised due to void volume⁷ and as pore size increases further, the specific surface area will eventually reduce to a level that will limit cell adhesion. Furthermore, this study has determined the optimal pore size for MC3T3-E1 pre-osteoblast activity. It has been hypothesised that the optimal pore size will vary with different cell types⁶ and another recent study from our laboratory has demonstrated that mesenchymal stem cells seeded on the smaller range of CG scaffolds and maintained in osteogenic culture for 3 weeks showed improved osteogenesis on the scaffolds with bigger pores²⁵. For this reason it is important to repeat this study with different cell types. However, regardless of these limitations, this paper has demonstrated that mean pore size does affect cell behavior within a scaffold and that subtle changes in pore size can have a significant effect on cell behavior. We also provide an insight into why the literature reports conflicting results on the optimal pore size required for bone tissue engineering, whereby increased specific surface area provided by scaffolds with small pores has a benefi- cial effect on initial cell attachment, but this is overcome by the improved cellular infiltration provided by scaffolds with larger pores suggesting that these scaffolds might be optimal for longer term in vitro culture with the aim of facilitating bone tissue repair.     

Initiation and growth characteristics of suspension cultures of Calendula officinalis cells

Callus cultures of marigold (Calendula officinalis L.) were induced on Murashige and Skoog medium with different concentrations of auxin (dichlorophenoxyacetic acid (2,4-D) or indole-3-acetic acid (IAA) and cytokinin (kinetin or 6-(,-dimethylallylamino)purine (2iP). Of all hormone combinations used in the medium, two were the most efficient in promoting callus development: 1.81 M (0.4 mg l^–1) 2,4-D and 1.85 M (0.4 mg l^–1) kinetin (0.4d–0.4k culture) or 0.45 M (0.1 mg l^–1) 2,4-D and 2.02 M (0.5 mg l^–1) 2iP (0.1d–0.5p culture). These combinations were selected to induce cell suspension cultures. The suspension cultures were maintained under light or dark conditions. The light stimulated cell aggregation in the cultures. In both cultures cells were undifferentiated under darkness, whereas in the light, rhyzogenesis was observed in 0.1d–0.5p culture. The cell growth and protein and oleanolic acid contents were determined. Initially, biomass production was similar under light and dark conditions, but after 7–8 months from the induction the cell growth was reduced by approximately 30% in the light, whereas the cell growth of the cultures maintained under darkness did not reveal any changes. The presence of oleanolic acid was detected in the suspension cultures kept in darkness. This compound reached two quantitative peaks: in the lag and stationary phases –- beyond the active growth phase of the culture cycle and its concentration was several times higher in 0.1d–0.5p culture than that in 0.4d–0.4k culture. It was for the first time that callus and suspension cultures were induced from the marigold plant.     

Biotransformation of theobromine to caffeine in suspension and polyurethane foam immobilized coffee (Coffea arabica L.) cells

Coffee (Coffea arabica L.) cells are capable of biotransforming theobromine to caffeine. In suspension culture of B2K medium, which is the production medium for caffeine, biotransformation was also more efficient than in DK medium. More caffeine was finally produced than calculated based on theobromine added to the medium. On the other hand, the efficiency of the biotransformation using immobilized cells in reticulate polyurethane foam cubes as a matrix varied with the phases. The biotransformation tended to be efficient under conditions which allowed the coffee cells to vigorously produce caffeine de novo.This paper is Part 75 in the series of Studies on Plant Tissue Cultures. For Part 74, see Kawaguchi K, Hirotani M, Furuya T, (1991) Phytochemistry, in press.     

Microtuberization of layered shoots and nodal cuttings of potato: The influence of growth regulators and incubation periods

A protocol is presented for the rapid induction of microtubers on micropropagated, layered potato shoots of Kennebec, Russet Burbank and Superior in medium devoid of growth regulators. Layered shoots microtuberized more rapidly and produced significantly larger microtubers compared with nodal cuttings. The addition of coumarin or (2-chloroethyl)-trimethylammonium chloride and benzyladenine to microtuberization medium, either had no effect or significantly reduced microtuber weight per shoots compared with medium containing only 80 g × 1^-1 sucrose and minimally affected the number of microtubers per shoot. Increasing the incubation period from 28 to 56 days did not affect the number but significantly increased the weight of microtubers per shoot and substantially increased the proportion, up to 20%, of microtubers heavier than 1 gram.Abbreviations Ba benzyladenine - ccc (2-chloroethyl) trimethylammonium chloride - coumarin 2h-1-benzopyran-2-one - ga₃ gibberellic acid     

Descriptive morphology of the reared eggs,larvae, and juveniles of the marine atherinid fish <Emphasis Type="Italic">Atherinomorus duodecimalis</Emphasis>

Embryonic, larval, and juvenile development of the marine atherinid Atherinomorus duodecimalis are described from laboratory-reared specimens. The eggs, measuring 1.15–1.24mm in diameter, were demersal and almost spherical in shape with numerous chorionic filaments. Hatching occurred 7 or 8 days after spawning, the newly hatched larvae measuring 4.5–5.1mm in body length (BL) and having 5+34=39 myomeres. Notochord flexion started at 6.9mm BL and finished at 8.3mm BL. Aggregate numbers of all fin rays were completed by 14mm BL. Eggs of this species could be distinguished from other known atherinid eggs on the basis of egg diameter and the arrangement, length, and number of chorionic filaments. Larvae were also distinctive in myomere counts, pigmentation, and locations of the anus and second dorsal fin origin.     

Kinetics of transient pump currents generated by the (H,K)-ATPase after an ATP concentration jump

Summary (H,K)-ATPase containing membranes from hog stomach were attached to black lipid membranes. Currents induced by an ATP concentration jump were recorded and analyzed. A sum of three exponentials ( ₁ ^-1 400 sec^–1, ₂ ^-1 100 sec^–1, ₃ ^-1 10 sec^–1; T = 300 K, pH 6, MgCl₂ 3 mm, no K⁺) was fitted to the transient signal. The dependence of the resulting time constants and the peak current on electrolyte composition, ATP conversion rate, temperature, and membrane conductivity was recorded. The results are consistent with a reaction scheme similar to that proposed by Albers and Post for the NaK-ATPase. Based on this model the following assignments were made: ₂ corresponds to ATP binding and exchange with caged ATP. ₁ describes the phosphorylation reaction E₁ · ATP E₁P. The third, slowest time constant ₃ is tentatively assigned to the E₁P E₂P transition. This is the first electrogenic step and is accelerated at high pH and by ATP via a low affinity binding site. The second electrogenic step is the transition from E₂K to E₁H. The E₂K E₁H equilibrium is influenced by potassium with an apparent K _0.5 of 3 mm and by the pH. Low pH and low potassium concentration stabilize the E₁ conformation.The authors wish to thank Dr. E. Grell and Mr. G. Schimmack. MPI Frankfurt, for synthesizing caged ATP, Mrs. S. Meister, Hoechst AG Frankfurt, for valuable help to prepare the (H,K)-ATPase, and Dr. W. Haase, MPI Frankfurt, for electron microscope pictures. (H,K)-ATPase for preliminary experiments was provided by Dr. W. Beil, Medizinische Hochschule Hannover, Dr. H. Swarts, University of Nijmegen, and Dr. G. Metzger, Hoechst AG Frankfurt. The work was supported by the Deutsche Forschungsgemeinschaft (SFB 169).     

Significance of rooting depth in mire plants: Evidence from natural <Superscript>15</Superscript>N abundance

Variation in stable nitrogen isotope ratios (¹⁵N) was assessed for plants comprising two wetland communities, a bog-fen system and a flood plain, in central Japan. ¹⁵N of 12 species from the bog-fen system and six species from the flood plain were remarkably variable, ranging from –5.9 to +1.1 and from +3.1 to +8.7, respectively. Phragmites australis exhibited the highest ¹⁵N value at both sites. Rooting depth also differed greatly with plant species, ranging from 5cm to over 200cm in the bog-fen system. There was a tendency for plants having deeper root systems to exhibit higher ¹⁵N values; plant ¹⁵N was positively associated with rooting depth. Moreover, an increasing gradient of peat ¹⁵N was found along with depth. This evidence, together with the fact that inorganic nitrogen was depleted under a deep-rooted Phragmites australis stand, strongly suggests that deep-rooted plants actually absorb nitrogen from the deep peat layer. Thus, we successfully demonstrated the diverse traits of nitrogen nutrition among mire plants using stable isotope analysis. The ecological significance of deep rooting in mire plants is that it enables those plants to monopolize nutrients in deep substratum layers. This advantage should compensate for any consequential structural and/or physiological costs. Good evidence of the benefits of deep rooting is provided by the fact that Phragmites australis dominates as a tall mire grass.     

Macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) resins—Versatile immobilization supports for biocatalysts

Crosslinked macroporous hydrophilic poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)s [abbreviated poly(GMA-co-EGDMA)] with identical chemical structure (60% of glycidyl methacrylate) but with varied average pore sizes (from 30 to 560 nm), specific surface areas (from 13.2 to 106.0 m²/g), specific volumes (from 0.755 to 1.191 cm³/g) and particle sizes (less than 100–650 μm) were synthesized via suspension polymerization. The influence of the resin properties on the loading of Candida antarctica lipase B (Cal-B) during immobilization and on the hydrolytic (hydrolysis of para-nitrophenyl acetate) and synthetic (ring-opening polymerization of -caprolactone) activity of the immobilized Cal-B were studied. Immobilization of Cal-B was performed at different temperatures and pH values. Cal-B immobilized at 30 °C and pH 6.8 was leading to increased activities. By decreasing the resin diameter: (i) the amount of Cal-B adsorbed onto the resin decreases, (ii) the conversion of para-nitrophenyl acetate increases (hydrolytic activity) and (iii) the conversion of -caprolactone and the molecular weight of the synthesized poly--caprolactone increases (synthetic activity). Varying the porosity parameters results in different hydrolytic and synthetic activities. Pore sizes of all synthesized resins (from 30 to 560 nm) are big enough to overcome diffusion limitations. Therefore increasing the pore size of the resins resulted in a large increase in the hydrolytic and synthetic activity. Increasing the specific surface area resulted in an increase of activities, as the result of alleviated substrate approach to the immobilized enzyme zones. The obtained results were compared to results from dried Cal-B powder and Novozyme 435. Resin with particle size less than 100 μm and pore size 48 nm had much higher hydrolytic activity than both dried Cal-B powder and Novozyme 435. Nearly similar trends were observed for the synthetic activity.Via the DMSO leaching technique we could show that about 80% of Cal-B was covalently attached to the macroporous resin.     

Packed-bed immobilized enzyme reactors for complex processes

Utilization of enzymic reactors for biotechnological-biomedical applications is currently developing at a sustained pace.Our present study concentrates on development of procedures for describing the performance of devices where enzyme-catalyzed reactions between two substrates take place, and for the rational design and optimization of the reactors considered. Within this context, an analytical model was developed for immobilized enzyme packed-bed reactors; it takes into account internal diffusion limitations for the cosubstrates, and hydrodynamic backmixing effects. In order to overcome the complex mathematical problems involved, the compartmental analysis approach was employed.Using this model, performance was simulated for various configurations of the enzymic unit, i.e. from a continuously operated stirred tank reactor (CSTR) to an essentially plug flow type. In addition, an experimental method is described for quantitatively assessing the backmixing effects prevailing in the reactor.The procedures established also provide the ground for further developments, particularly for systems where, in parallel to the enzymic reaction, additional processes (e. g. complexation) take place.List of Symbols C _j,i mM Concentration of substrate j in the pores of stage - iD _j cm²/s Internal (pore) diffusion coefficient of substrate j; defined in Eq. (7) - D _e cm²/s Axial dispersion diffusion coefficient - D _j, cm²/s cm²/s Bulk diffusion coefficient for substrate j - E mM Enzyme concentration inside the catalytic pores - J _j,immol/s/cm² Net flux of substrate j taking place from the bulk of stage i into the corresponding pores; defined in Eq. (6) - K _m,1, K _m,2 mM Michaelis-Menten constants for cosubstrates 1 and 2, respectively - k s ^–1 Catalytic constant - k _s cm/s Catalytic constant - n Total number of elementary stages in the reactor - Q cm³/s Volumetric flow rate throught the reactor - r cm Radius of the pore - R _j,i mM/s Reaction rate of substrate j in stage i, in terms of volumetric units - S cm² Internal surface of a pore - S _j,0 mM Concentration of substrate j in the reactor feed - S _j,i–1, S _j,i mM Concentration of substrate j in the bulk phase leaving stages i — 1 and i, respectivley - V _i cm³ Total volume of stage i (bulk phase + pore phase + inert solid carrier) - V cm³ Total volume of the reactor - V _m ^* mmol/s/cm² Maximal reaction rate in terms of surface units; defined in Eq. (8) - V _m mM/s Maximal reaction rate in terms of volumetric units; defined in Eq. (8) - V _p cm³ Volume of one pore - y cm Axial coordinate of the pores - y ₀ cm Depth of the pores - Z cm Axial coordinate of the reactor - Z ₀ cm Length of the reactor - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - Ratio between the radius of the enzyme molecule and the radius of the pore (dimensionless) - V₁ Dimensionless parameter; defined in Eq. (21) - v₂ Dimensionless parameter; defined in Eq. (21) - Q Volumetric packing density of catalytic particles (dimensionless) - Ø Porosity of the catalytic particles (dimensionless) - Ø Dimensionless concentration of substrate j in pores of stage i; defined in Eq. (16) - _j,i-1,_j,i Dimensionless concentration of substrate j in the bulk phase of stage i; defined in Eq. (18) - Dimensionless position; defined in Eq. (16) - ² s² Variance; defined in Eq. (33) - Mean residence time in the reactor; defined in Eq. (33)     

Influence of arbuscular mycorrhizal fungi on soil structure and soil water characteristics of vertisols

The influence of inoculation with arbuscular mycorrhizal fungi (AM fungi) on soil water characteristics of fast and slowly wetted vertisol samples was studied. Vertisols characteristically have a low stability to wetting, and the disruption of their larger pores when they swell leads to reduced water infiltration and thereby to runoff. The degree of aggregate breakdown determines the ability of the soil to drain. A vertisol was used in this pot experiment with four treatments: T₁: Pasteurized soil, T₃: Pasteurized soil, with plants, T₄: Inoculated, pasteurized soil, with plants, T₅: Unpasteurized soil, with plants. A treatment using inoculated, pasteurized soil (T₂) was included in a related study (Bearden and Petersen, 2000) comparing aggregate stability, and the present study follows the same numbering to aid in comparison of experiments. After fast, disruptive wetting, the soil inoculated with AM fungi (T₄) was found to have a lower soil water content than did the soils from the other treatments at matric potentials lower than –3.92 kPa. This indicates greater drainage from pores smaller than 75 m for the soil inoculated with AM fungi, and the greater drainage appears to be directly related to a characteristic pore range between 67 and 75 m. The soil without plants (T₁), when wetted fast, had a lower soil water content at matric potentials higher than –3.92 kPa than soils from the other treatments, which indicates less pore volume due to pores larger than 75 m in the treatment without plants. The pore indexes, calculated as the ratio between the slope of the fast and the slope of the slowly-wetted water characteristics, generally had the highest values for the soil inoculated with AM fungi (T₄) from matric potential 0.00 to –0.29 kPa. In this matric potential range, the pore indexes were less than one. The unpasteurized soil with naturally present AM fungi (T₅) generally had the highest pore indexes from matric potential –0.49 to –3.92 kPa, and the pore indexes in this matric potential range were above one. These results indicate the smallest loss of very large pores in the soil inoculated with AM fungi (T₄) and the largest gain of smaller sized pores in the unpasteurized soil (T₅). This suggests that the resistance to breakdown of the largest pores is related to the presence of roots, and that the gain of groups of smaller pores is related to the presence of hyphae.     

Heterogeneous Long Chain Acyl-CoA Synthetases Control Distribution of Individual Fatty Acids in Newly-Formed Glycerolipids of Neuronal Cells Undergoing Neurite Outgrowth

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growth factor (NGF) were labeled with [³H]glycerol, [³H]oleic acid (OA) or [³H]arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin. Triacsin C (4.8 M), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous [³H]OA into glycerolipids by 30–90%, and increased by about 60% the accumulation of free [³H]OA in the cells. However it did not affect the incorporation of endogenous fatty acids nor of exogenous [³H]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 M, respectively; and in cytoplasm, 6 and 0.6 nmol/ min/mg protein and 38 and 60 M, respectively. Triacsin C (2–33 M) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC₅₀ and apparent Ki values were 13.5 M and 14 M in microsomes, and 3.8 M and 4 M in cytoplasm. NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids.     

Cell-to-cell diffusion selectivity in staminal hairs ofSetcreasea purpurea

Summary Diffusion coefficients for FITC-molecular probes in intercellular pores (D) and rate of molecular probe loss into the vacuole (k₁) have been obtained for FITC molecular probes in staminal hairs ofSetcreasea purpurea. The kinetic curves of FITC-Gly, -Ala, -Leu,-Ser, -Thr, -Cys, -Met, -Tyr, -Asp, -Glu, -Asn, -Gln, -Lys, -His,-Arg, -(Asp)₂, -(Glu)₂, -(Lys)₂, -(Asp)₃, -(Glu)₃, -(Gln)₂, -(Gln)₃, -(Gln)₄, and carboxyfluorescein (group I probes) matched the curves calculated for simple diffusion through a chain of cells, while the majority of kinetic curves of FITC-Phe, and -Try (group II probes) did not. None of the kinetic curves for FITC-(Met)₂ and -(His)₂ (group III probes) matched. Average Ds for group I probes ranged from 0.77× 10^–8cm²/s to 3.75× 10^–8cm²/s and for group II probes were 0.50× 10^–8cm²/s. A meaningful average D for group III probes could not be calculated. Average k₁ for group I probes ranged from 1.62× 10^–7/m²/s to 13.21× 10^–7/m²/s, and for group II probes were 5.42 and 11.54× 10^–7/m²/s. Average k₁s for group III probes could not be calculated. Symplastic transport occurred by cell-to-cell diffusion for most of the probes (e.g., group I probes) but not always for some (e.g., group II probes) and never for others (group III probes). The rate of cell-to-cell diffusion and loss within the vacuole depended upon the molecule's specific structure, molecular weight and charge. We concluded that plasmodesmata select for molecules that are hydrophilic, small and have a charge of from — 2 to — 4, and against molecules that contain either Phe, Try, Met or His groups.Abbreviations CF carboxyfluorescein - D diffusion coefficients for FITC-molecular probes in intercellular pores - k₁ rate of FITC-molecular probe loss     

Effects of hypoxia on the pulmonary microvascular permeability in adult rabbit lung

Within the last 30 years, researchers have explored what role hypoxia might play in causing permeability changes in the pulmonary microvasculature. Since the data accumulated thus far are unclear, the effects of hypoxia on microvascular transport in the isolated, Ringer's perfused adult rabbit lung was observed and the following parameters were measured or computed for both oxygenated and hypoxic perfusates: pulmonary arterial (r_a) and pulmonary venous (r_v) resistances, pulmonary capillary filtration coefficients (K_f), and pulmonary capillary endothelial reflection coefficients () for NaCl and inulin. Separate reservoir bottles were used to create the desired oxygenated (aeration of solution with 95% O₂-5% CO₂) gas mixture or hypoxic (aeration of solution with 95% N₂-5% CO₂) gas mixture. A higher, but not significant, resistance value was found during the oxygenated state. A significant increase in the pulmonary capillary filtration coefficient during hypoxia (10.72 × 10^–4±0.446 × 10^–4 cm³/s cm H₂O for the hypoxic perfusate and 8.80 × 10^–4±0.384 × 10^–4 cm³/s cm H₂O for the oxygenated perfusate) was found and a significant difference between oxygenated and hypoxic pulmonary capillary reflection coefficients for inulin was computed (oxygenated solution revealed a finding of 0.120±0.003 and the hypoxic solution revealed 0.105±0.002). These findings imply a change in the microvascular permeability during hypoxia. According to the pore theory, a change in pore number, pore size, or both could have occurred. However, from the reflection coefficient data, a change in pore radius seems most likely.