A new continuous cell line from larval ovaries of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A new continuous cell line from ovarian tissue of commercial variety “Kolar Gold” of silkworm, Bombyx mori, was established and designated as DZNU-Bm-12. The tissue was grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat-inactivated B. mori hemolymph at 25 ± 1°C. The migration of partially attached small round refractive cells from the fragments of ovarioles began from the beginning of explantation. The cells multiplied partially attached in the primary culture initially, and some of them become freely suspended after 20 passages. The cells were adapted to MGM-448 and TNM-FH media each with 10% FBS and the population doubling time of cell line was about 36 and 24 hr, respectively. The chromosome number was near diploid at initial passages and slightly increased at 176th passage, but a few tetraploids and hexaploids were also observed. DNA profiles using simple sequence repeat loci established the differences between DZNU-Bm-12 and DZNU-Bm-1 and most widely used Bm-5 and BmN cell lines. The cell line was found susceptible to B. mori nucleopolyhedrovirus (BmNPV) with 85–90% of the cells harboring BmNPV and having an average of 3–17 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV-based baculoviral expression system and also for studying in vitro virus replication.     

Expression and characterization of a recombinant Drosophila tyramine-β-hydroxylase in silkworm infected with recombinant baculovirus

The insect nervous system contains biogenic amines such octopamine (OA), which is synthesized from tyramine (TA) by catalysis of tyramine-β-hydroxylase (TβH). In this study, the Drosophila 70 kDa tyramine-β-hydroxylase (DmTβH) protein was purified after the recombinant nucleopolyhedrovirus isolated from Bombyx mori (BmNPV) containing the TβH gene was injected into the hemocoel of the fifth instar larvae from the d17 B. mori strain. Western blot analysis revealed an immunoreactive band with a molecular mass of 70 kDa. The products formed by incubating the enzyme reaction mixture were separated and detected by reverse phase high-performance liquid chromatography. The optimum pH, temperature, and incubation time for the conversion of TA to OA were 7.6, 25 °C, and 30 min, respectively. The inhibitory experiments using various concentrations of 1-(2-methoxy-5-methylphenyl) imidazole-2(3H)-thione (MMIT) showed that MMIT inhibited DmTβH dose-dependently and that this method can be applied for screening DmTβH inhibitors.     

Serum-free culture of an embryonic cell line from <Emphasis Type="Italic">Bombyx mori</Emphasis> and reinforcement of susceptibility of a recombinant BmNPV by cooling

We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67 ± 5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.     

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients

Background aimsAlloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use.MethodsWe describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells.ResultsNK cells (6.0 ± 1.2 × 10⁸) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR⁺ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML.ConclusionsThe approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.     

Formulation of Pseudomonas chlororaphis strains for improved shelf life

Formulations of Pseudomonas strains with long-term shelf life are needed for commercial use in biological disease control and growth promotion in crops. In the present work Pseudomonas chlororaphis (Pc) 63-28 formulated with coconut fiber [moisture content (MC) of 80%], talc (MC 8%) or peat (MC 40%), with or without the addition of carboxymethylcellulose or xanthan gum, and formulations of Pc 63-28 and P. chlororaphis TX-1 in coconut fiber with water contents (v:v) of 75%, 45%, and 25%, were evaluated in terms of shelf life and cell viability. The shelf life of Pc 63-28 was longer when formulated in coconut fibre with a MC was 80% than in the other formulations and longer at 3 ± 1 °C compared to 22 ± 1 °C. Densities of viable Pc 63-28 cells in coconut fiber stored at 3 ± 1 °C did not decline significantly during 224 days when the MC was 80% and within 120 days at 75% MC. Densities of Pc TX-1 in coconut fiber of 75% MC did not decline within 60 days at 3 ± 1 °C. P. chlororaphis 63-28 survived longer in deionized water and buffer than in canola oil. Cells of Pc 63-28 cells formulated in coconut fibre of 80% MC after storage for 140 days at 3 ± 1 °C in coconut fiber improved hydroponic growth of hydroponic lettuce and better than cells freshly recovered from culture. We conclude that coconut fiber is a carrier of superior performance in maintaining shelf life of Pseudomonas strains. The observed shelf life would be sufficient for practical use of Pseudomonas strains as tools for disease control and growth promotion in crops.     

Behavioral thermoregulation and critical thermal limits of giant keyhole limpet Megathura crenulata (Sowerby 1825) (Mollusca; Vetigastropoda)

The thermoregulatory behavior of the giant keyhole limpet Megathura crenulata was determined in a horizontal thermal gradient during the day at 18.9 °C and 18.3 °C for the night. The final preferendum determined for giant keyhole limpets was of 18.6±1.2 °C.Limpets' displacement velocity was 10.0±3.9 cm h⁻¹ during the light phase and 8.4±1.6 cm h⁻¹ during the dark phase. The thermotolerance (measured as CTMax at 50%) was determined in a keyhole limpet in three acclimation temperatures 17, 20, and 23 °C. Limpets were subjected to water increasing temperatures at a rate of 1 °C every 30 min, until they detached from the substrate. The critical thermal maximum at 50% was 27.2, 27.9 and 28.3 °C respectively.     

A reduced core to skin temperature gradient,not a critical core temperature,affects aerobic capacity in the heat

The purpose of this study was to determine the impact of the core to skin temperature gradient during incremental running to volitional fatigue across varying environmental conditions. A secondary aim was to determine if a “critical” core temperature would dictate volitional fatigue during running in the heat. 60 participants (n=49 male, n=11 female; 24±5 yrs, 177±11 cm, 75±13 kg) completed the study. Participants were uniformly stratified into a specific exercise temperature group (18 °C, 26 °C, 34 °C, or 42 °C) based on a 3-mile run performance. Participants were equipped with core and chest skin temperature sensors and a heart rate monitor, entered an environmental chamber (18 °C, 26 °C, 34 °C, or 42 °C), and rested in the seated position for 10 min before performing a walk/run to volitional exhaustion. Initial treadmill speed was 3.2 km h⁻¹ with a 0% grade. Every 3 min, starting with speed, speed and grade increased in an alternating pattern (speed increased by 0.805 km h⁻¹, grade increased by 0.5%). Time to volitional fatigue was longer for the 18 °C and 26 °C group compared to the 42 °C group, (58.1±9.3 and 62.6±6.5 min vs. 51.3±8.3 min, respectively, p<0.05). At the half-way point and finish, the core to skin gradient for the 18 °C and 26 °C groups was larger compared to 42 °C group (halfway: 2.6±0.7 and 2.0±0.6 vs. 1.3±0.5 for the 18 °C, 26 °C and 42 °C groups, respectively; finish: 3.3±0.7 and 3.5±1.1 vs. 2.1±0.9 for the 26 °C, 34 °C, and 42 °C groups, respectively, p<0.05). Sweat rate was lower in the 18 °C group compared to the 26 °C, 34 °C, and 42 °C groups, 3.6±1.3 vs. 7.2±3.0, 7.1±2.0, and 7.6±1.7 g m⁻² min⁻¹, respectively, p<0.05. There were no group differences in core temperature and heart rate response during the exercise trials. The current data demonstrate a 13% and 22% longer run time to exhaustion for the 18 °C and 26 °C group, respectively, compared to the 42 °C group despite no differences in beginning and ending core temperatures or baseline 3-mile run time. This capacity difference appears to result from a magnified core to skin gradient via an environmental temperature advantageous to convective heat loss, and in part from an increased sweat rate.     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Toxin production,retention, and extracellular release by Dinophysis acuminata during extended stationary phase and culture decline

Dinophysis spp. produce diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins. The extent to which the dinoflagellate cells retain their toxicity in stationary phase, a period when cells are most toxic, and their transition into cell death is not known. Here we present results on the production, recycling, retention, and release of toxins from a monoculture of Dinophysis acuminata during these two important stages. Once stationary phase was reached, cultures were divided between light and dark treatments to identify if light influenced toxin dynamics. Light was required for long-term cell maintenance (>2 months) of D. acuminata in the absence of prey, however, in the dark, cells in stationary phase survived on reserves alone for four weeks before beginning to decline. Cells maintained relatively constant levels of intracellular OA (0.39 ± 0.03 pg/cell, 0.44 ± 0.05 pg/cell), DTX1 (0.45 ± 0.09 pg/cell, 0.64 ± 0.10 pg/cell) and PTX2 (10.4 ± 1.4 pg/cell, 11.0 ± 1.9 pg/cell) in the dark and light treatments, respectively, throughout stationary phase and into culture decline. Toxin production was only apparent during late exponential and early stationary growth when cells were actively dividing. In general, the concentration of dissolved (extracellular) toxin in the medium significantly increased upon culture aging and decline; cells did not appear to be actively or passively releasing toxin during stationary phase, but rather extracellular release was likely a result of cell death. Light availability did not have an apparent effect on toxin production, quotas, or intracellular vs. extracellular distribution. Together these results suggest that a bloom of D. acuminata would retain its cellular toxicity or potency as long as the population is viable, and that cells under conditions of low light (e.g., at the boundary or below euphotic zone) and/or minimal prey could maintain toxicity for extended periods.     

Biology and thermal requirements of Chrysoperla genanigra (Neuroptera: Chrysopidae) reared on Sitotroga cerealella (Lepidoptera: Gelechiidae) eggs

Chrysoperla genanigra Freitas is a common green lacewing associated with melon pests in the Northeastern Brazil. All life stages of this recently described species were studied under a range of constant temperature conditions (17, 21, 25, 29, 33, 35 and 37 °C), a photoperiod of 12 h:12 h (L:D) and 70 ± 10% relative humidity. Adults of C. genanigra were fed on a diet consisting of a 1:1 (v/v) mixture of brewer’s yeast and honey, while larvae were provided with eggs of Sitotroga cerealella (Olivier) ad libitum. The duration of preimaginal development of the species was inversely proportional to temperature and ranged from approximately 63 days at 17 °C to 15 days at 35 °C. The percentage of adult emergence varied from 6.7% at 17 °C to 76.7% at 25 °C, although no larvae were able to complete development at 37 °C. The lower thermal threshold for total preimaginal development was approximately 10.8 °C and the thermal requirement was 336.7 degree-days. Egg production, along with the longevity of both males and females, were significantly affected by temperature. It is concluded that the best temperature for rearing C. genanigra is 25 °C, with the lowest preimaginal mortality and the highest egg production (992.7 eggs/female).     

Cool gleaners: Thermoregulation in sympatric bat species

Thermoregulatory behavior in temperate bats is influenced by gender, food availability, ambient temperature and reproduction. Ecologically and morphologically similar bat species (Myotis bechsteinii, M. nattereri, and Plecotus auritus; Vespertilionidae) facing similar diurnal conditions should therefore not differ in their thermoregulatory behavior. Identified day roosts (n = 23) of radio-tagged bats (n = 30) were spread over an area of 33.1 ha, but ambient temperature did not differ between roosting sites. Furthermore, there was no significant difference in cardinal direction, roost height, canopy coverage, and breast height diameter between day roosts used by the three species. Minimum roost temperatures and isolation values, however, differed significantly between our species with lowest values in P. auritus. The range of skin temperatures (min–max) recorded by temperature-sensitive transmitters was not species-specific with the lowest ranges in late pregnancy (mean ± SD: 7.1 ± 1.1 °C) and highest in post-lactation (mean ± SD: 13.1 ± 1.1 °C). The minimum skin temperature, however, was species-specific with the lowest values in P. auritus (mean ± SD: 20.2 ± 1.1 °C), intermediate in M. nattereri (mean ± SD: 23.4 ± 1.0 °C), and the highest in M. bechsteinii (mean ± SD: 26.8 ± 1.0 °C). Species-specific usage of energy-saving mechanisms might represent an important niche differentiation of species. Different mechanisms might allow, e.g. one species to occupy colder roosts with higher temperature variations or to shorten foraging times due to distinct thermoregulatory behavior.     

Rotator cuff tendinopathy alters the muscle activity onset and kinematics of scapula

Athletes with rotator cuff (RC) tendinopathy demonstrate an aberrant pattern of scapular motion which might relate to deficits in the scapular muscles. This study aimed to determine whether alteration in scapular kinematics is associated with deficits in the activity onset of scapular muscles. Forty-three male volleyball players (17 asymptomatic and 26 with RC tendinopathy) joined the study. Three-dimensional scapular kinematics was quantified using an acromial marker cluster method. The activity onset of the upper (UT), middle (MT), and lower trapezius (LT), and serratus anterior (SA) during arm abduction was assessed with electromyography. Athletes with RC tendinopathy demonstrated less scapular upward rotation (6.6 ± 2.3 vs. 8.2 ± 1.1°, p = 0.021) in the early phase of shoulder abduction from 0° to 30° when compared to asymptomatic athletes. The tendinopathy group had delayed activity onset of LT (14.1 ± 31.4 ms vs. 74.4 ± 45.1 ms, p < 0.001) and SA (−44.9 ± 26.0 ms vs. 23.0 ± 25.2 ms, p < 0.001) relative to UT when compared to the asymptomatic group. In asymptomatic athletes, earlier activity onset of MT and LT relative to UT was associated with more scapular upward rotation during 0–30° of abduction (r = 0.665, p = 0.021) and 30–60° of abduction (r = 0.680, p = 0.015), respectively. Our findings showed the control of the scapular upward rotation is related to the activity onset of the scapular muscles in athletes.     

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me₂SO). Me₂SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me₂SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me₂SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.     

Characterization and expression of the pseudorabies virus (NYJ strain) glycoproteins in Bombyx mori cells and larvae

To characterize the NYJ strain of pseudorabies virus (PRV; Alphaherpesvirus of swine) isolated from the serum of an infected swine in Korea, the nucleotide sequence of three major glycoproteins (gB, gC, and gD) was analyzed. The expression of most potent immunogenic glycoprotein (gD) was also investigated using a Bombyx mori nucleopolyhedrovirus (BmNPV) expression system. The length of the glycoprotein genes corresponding to gB, gC, and gD of the NYJ strain were 2751 bp, 1443 bp, and 1203, respectively, and their identity ranged from 94.2% to 99.8% when compared with other strains. Phylogenetic analyses using these sequences showed that the NYJ strain forms a distinct branch with high bootstrap support. A novel transfer vector (pBmKSK4) was engineered with the polyhedrin promoter of BmNPV and a 6xHis tag to express glycoprotein gD in Bm5 cells and silkworm, B. mori, larvae. The immunogenicity of recombinant gD was demonstrated by its specific detection in both Bm5 cells and silkworm larvae by porcine anti-PRV antibody. The results of this study have implications both for the taxonomy of Korean PRV strains and vaccine development.     

Effect of the timing of ice slurry ingestion for precooling on endurance exercise capacity in a warm environment

It has been demonstrated that precooling with ice slurry ingestion enhances endurance exercise capacity in the heat. However, no studies have yet evaluated the optimal timing of ice slurry ingestion for precooling. This study aimed to investigate the effects of varying the timing of ice slurry ingestion for precooling on endurance exercise capacity in a warm environment. Ten active male participants completed 3 experimental cycling trials to exhaustion at 55% peak power output (PPO) after 15 min of warm-up at 30% PPO at 30 °C and 80% relative humidity. Three experimental conditions were set: no ice slurry ingestion (CON), pre-warm-up ice slurry ingestion (−1 °C; 7.5 g kg⁻¹) (PRE), and post-warm-up ice slurry ingestion (POST). Rectal and mean skin temperatures at the beginning of exercise in the POST condition (37.1±0.2 °C, 33.8±0.9 °C, respectively) were lower than those in the CON (37.5±0.3 °C; P<0.001, 34.8±0.8 °C; P<0.01, respectively) and PRE (37.4±0.2 °C; P<0.01, 34.6±0.7 °C; P<0.01, respectively) conditions. These reductions increased heat storage capacity and resulted in improved exercise capacity in the POST condition (60.2±8.7 min) compared to that in the CON (52.0±11.9 min; effect size [ES]=0.78) and PRE (56.9±10.4 min; ES=0.34) conditions. Ice slurry ingestion after warm-up effectively reduced both rectal and skin temperatures and increased cycling time to exhaustion in a warm environment. Timing ice slurry ingestion to occur after warm-up may be effective for precooling in a warm environment.     

Thermal tolerance,growth and oxygen consumption of Labeo rohita fry (Hamilton, 1822) acclimated to four temperatures

A 30 day feeding trial was conducted using a freshwater fish, Labeo rohita (rohu), to determine their thermal tolerance, oxygen consumption and optimum temperature for growth. Four hundred and sixteen L. rohita fry (10 days old, 0.385±0.003 g) were equally distributed between four treatments (26, 31, 33 and 36 °C) each with four replicates for 30 days. Highest body weight gain and lowest feed conversion ratio (FCR) was recorded between 31 and 33 °C. The highest specific growth rate was recorded at 31 °C followed by 33 and 26 °C and the lowest was at 36 °C. Thermal tolerance and oxygen consumption studies were carried out after completion of growth study to determine tolerance level and metabolic activity at four different acclimation temperatures. Oxygen consumption rate increased significantly with increasing acclimation temperature. Preferred temperature decided from relationship between acclimation temperature and Q₁₀ values were between 33 and 36 °C, which gives a better understanding of optimum temperature for growth of L. rohita. Critical thermal maxima (CTMax) and critical thermal minima (CTMin) were 42.33±0.07, 44.81±0.07, 45.35±0.06, 45.60±0.03 and 12.00±0.08, 12.46±0.04, 13.80±0.10, 14.43±0.06, respectively, and increased significantly with increasing acclimation temperatures (26, 31, 33 and 36 °C). Survival (%) was similar in all groups indicating that temperature range of 26–36 °C is not fatal to L. rohita fry. The optimum temperature range for growth was 31–33 °C and for Q₁₀ values was 33–36 °C.     

Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties

An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me₂SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume (V_b) and the permeability coefficient of water (L_p) for RBL cells are determined to be 41% [n = 18, correlation coefficient (r²) of 0.903] of original/isotonic volume, and 0.32 ± 0.05 μm/min/atm (n = 8, r² > 0.963), respectively, for room temperature (22 °C). From experiment (2), the permeability coefficient of water (L_p) and of Me₂SO (P_s) for RBL cells are 0.38 ± 0.09 μm/min/atm and (0.49 ± 0.13) × 10⁻³ cm/min (n = 5, r² > 0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell osmotic response and determine cell membrane transport properties; and (4) reduce labor requirements for its disposability and ensure low manufacturing costs.     

The silk cocoon of the silkworm,Bombyx mori: Macro structure and its influence on transmural diffusion of oxygen and water vapor

The cocoon of insect larvae is thought to help conserve water while affording mechanical protection. If the cocoon is a barrier to water loss, then it must also impose a barrier to inward oxygen diffusion. We tested this hypothesis in pupae of the silkworm, Bombyx mori. The rate of water loss and oxygen uptake (V?O₂) at 25 °C was measured in control pupae in their naturally spun cocoon and in exposed pupae experimentally removed from their cocoon. Additional measurements included the oxygen diffusion coefficient, DO₂, of the cocoon wall and dimensions and density of the cocoon fibers. Water loss (as % body mass loss) in both control and exposed pupae was ～ 1%^.day^? 1, and was not significantly different between populations. Similarly, V?O₂ was statistically identical in both control and exposed pupae, at 0.22 ± 0.01 and 0.21 ± 0.02 mL g^? 1 · h^? 1, respectively. The silk fiber diameter was significantly different in the outer fibers, 26 ± 1 µm, compared with 16 ± 1 µm for the inner fibers lining the cocoon. Inner fibers were also spun significantly more densely (20.8 ± 1.2 mm^? 1 transect) than outer fibers (8.3 ± 0.2). Mean DO₂ at 25 °C was 0.298 ± 0.002 cm² · s^? 1, approximately the same as unstirred air. These data indicate that the cocoon, while creating a tough barrier offering mechanical protection to the pupa, imposes no barrier to the diffusion of oxygen or water vapor.     

Novel dammarane saponins from Gynostemma pentaphyllum and their cytotoxic activities against HepG2 cells

Two new dammarane saponins, 2α,3β,12β-trihydroxydammar-20(22),24-diene-3-O-[β-d-glucopyranoxyl(1→2)-β-d-6″-O-acetylglucopyranoside (1, namely damulin C) and 2α,3β,12β-trihydroxydammar-20(21),24-diene-3-O-[β-d-glucopyranoxyl(1→2)-β-d-6″-O-acetylglucopyranoside (2, namely damulin D), were isolated from the ethanol extract of Gynostemma pentaphyllum, which had been heat processed by steaming at 125 °C. The NMR spectroscopic data of the novel saponins were completely assigned by using a combination of 2D NMR experiments including ¹H–¹H COSY, HSQC, and HMBC. Their cytotoxic activities of human liver adenocarcinoma HepG2 cells were evaluated in vitro. They showed cytotoxicities against HepG2 cell line with IC₅₀ of 40 ± 0.7 and 38 ± 0.5 μg/ml, respectively.