Baculovirus production for gene therapy: the role of cell density,multiplicity of infection and medium exchange

One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used, with a high MOI “delaying” the drop in production to higher cell densities. Medium replacement at the time of infection using a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers as high as 2.6 × 10¹⁰ IP.mL⁻¹ were obtained in cultures infected at 3.5 × 10⁶ cells.mL⁻¹, while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange.     

Banking of osteochondral allografts. Part I. Viability assays adapted for osteochondrol and cartilage studies

The aim of this study was to adapt a reliable, reproducible and simple viability assay for cartilage and osteochondral studies. The previous assays (radioisotope uptake, assessment of matrix components, histological methods, oxygen consumption etc.) were complex, laborious, time consuming or suffer from difficulty of interpretation. MTT assay was chosen because it has been widely and successfully used in different cell and tissue studies, but has not been published on human solid articular cartilage. Fresh intact cartilage samples of human tali were tested to investigate the assay. The reliability of the MTT assay was also tested by an fluorescent dye combination. The MTT assay is based on the production of purple formazan pigment from methyltetrazolium salt by the mitochondrial enzymes of viable chondrocytes. The enzyme kinetics of the reaction was also investigated because it was unknown in the case of cartilage. The amount of pigment formed can be measured by spectrophotometry after extraction by methyl cellosolve. The color density is proportional to mitochondrial enzyme activity, reflecting the number of viable chondrocytes. The optimal reagent concentration, biopsy size, and incubation period were established. There is a linear relationship between the cartilage weight and the pigment production activity. A 9.8% nonspecific raction was observed in the negative controls. The enzyme kinetics of the reaction was also investigated. The MTT clevage up to 0.1% (w/v) follows the Michaelis kinetics. We calculated the Michaelis constant (2835 ± 130 μM), the maximal velocity (36 ± 3.2 × 10⁻⁵μMsec⁻¹) and the velocity constant (1.27 ± 0.2 × 10⁻⁷sec⁻¹) of the reaction. The latter is a significant marker for each tissue type. The viability of cartilage was also assessed and calculated by a fluorescent dye combination comprising 1 μg/ml propidium iodide (PI) and 4 μM/ml SYTO-16 stains. The PI stains dead cells (red fluorescence), the SYTO-16 stains live cells (green fluorescence). The staining can be visualised simultaneously, and the live/dead ratio can be calculated by image analysis software from saved image files. The MTT assay is a simple, non-expensive, efficient, reliable, reproducible, sensitive viability test for cartilage studies. The MTT reduction assay and the staining method were corrobative. This revised version was published online in July 2006 with corrections to the Cover Date.     

Toxicity of dodecylpyridinium and cetylpyridinium clorides against phosphate-accumulating bacterium

The antibacterial effect of cationic surfactants against the pure culture of phosphate (P)-accumulating bacterium Acinetobacter junii was investigated. The estimated EC₅₀ values of the N-dodecylpyridinium chloride (DPC) for growth inhibition was 1.4±0.5 × 10⁻⁶ mol L⁻¹ and for the inhibition of the P-uptake rates 7.3±2.6 × 10⁻⁵ mol L⁻¹. The estimated EC₅₀ values of the N-cetylpyridinium chloride (CPC) for growth inhibition was 4.9±1.3 × 10⁻⁷ mol L⁻¹ and for the inhibition of the P-uptake rates 7.7±2.9 × 10⁻⁶ mol L⁻¹. This suggests the importance of controlling the amounts of cationic surfactants in influent of the wastewater treatment systems in order to avoid the possible failure of the biological P removal from wastewaters.     

In Vitro Culture of Rudimentary Embryos of Ilex paraguariensis: Responses to Exogenous Cytokinins

The effects of benzyladenine (BAP), kinetin (KIN), zeatin (ZEA), isopentenyladenine (2iP), and thidiazuron (TDZ) were studied on in vitro growth of rudimentary embryos of Ilex paraguariensis St. Hil. Heart stage zygotic embryos were removed from seeds of immature, light green fruits and cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without three concentrations of BAP, KIN, ZEA, 2iP, or TDZ. Cultures were incubated in darkness at 27 ± 2°C. Media containing 4.4 × 10⁻⁶ m BAP, 4.6 × 10⁻⁶ m KIN, or 4.9 × 10⁻⁶ m 2iP were totally ineffective in inducing embryo growth after culture for 28 days. However, lower concentrations of these compounds (4.4 × 10⁻⁸ m BAP, 4.6 × 10⁻⁸ m KIN, 4.5 × 10⁻⁸ m ZEA, or 4.9 × 10⁻⁸ m 2iP) promoted embryo growth. TDZ at 9.9 × 10⁻⁹ m, 9.9 × 10⁻⁸ m, or 9.9 × 10⁻⁷ m induced embryo growth at similar rates. The maximum percentage of embryos converted to seedlings was achieved when the medium was supplemented with 4.5 × 10⁻⁷ m ZEA. Received August 1, 1997; accepted February 19, 1998     

Toxicity of anionic and cationic surfactant to Acinetobacter junii in pure culture

The harmful effects of surfactants to the environment are well known. We were interested in investigating their potential toxicity in a pure culture of Acinetobacter junii, a phosphate (P)-accumulating bacterium. Results showed a high acute toxicity of sodium dodecyl sulfate (SDS) and hexadecyltrimethylammonium bromide (HDTMA) against A. junii. The estimated EC₅₀ values of the HDTMA for the inhibition of CFUs in the pure culture of A. junii was 3.27 ± 1.12 × 10⁻⁷ mol L⁻¹ and for the inhibition of the P-uptake rates 2.47 ± 0.51 × 10⁻⁶ mol L⁻¹. For SDS, estimated EC₅₀ values for the inhibition of CFUs in the pure culture of A. junii was 5.00 ± 2.95 × 10⁻⁶ mol L⁻¹ and for the inhibition of the P-uptake rates 3.33 ± 0.96 × 10⁻⁴ mol L⁻¹. The obtained EC₅₀ values in the standardised yeast toxicity test using Saccharomyces cerevisiae were 3.03 ± 0.38 × 10⁻⁴ and 4.33 ± 0.32 × 10⁻⁵ mol L⁻¹ for SDS and HDTMA, respectively. These results emphasized the need to control concentrations of surfactants entering the activated sludge system. The negative effects of these toxicants could greatly decrease populations of P-accumulating bacteria, as well as eukaryotic organisms, inhabiting activated sludge systems, which in turn could result in the decrease of the system efficiency.     

Rapid titer assay of adenovirus containing green fluorescent protein gene using flow cytometric analysis

The plaque assay has been widely used for titration of adenovirus (AdV). However, it takes usually 2-3 weeks, so this slow assay often impedes bioprocess development of large-scale AdV production. In this study, we developed a rapid AdV titration assay that can be done within a day. Further, unlike the plaque assay, this assay does not require a laborious serial dilution of samples. This rapid assay can be achieved by using green fluorescent protein (GFP) as a marker gene and flow cytometric analysis. It yields a good correlation between infectious titer of AdV harboring GFP and flow cytometric parameters such as average green fluorescence intensity or % of infected cells. Taken together, this rapid assay will facilitate bioprocess development for efficient large-scale AdV production.     

Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7×10⁻⁶. In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F₁ generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P₀ generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism. ^★Equal contributions to this article.     

Transdermal delivery of diclofenac sodium through rat skin from various formulations

The aim of this study was to evaluate and compare the in vitro and in vivo transdermal potential of w/o microemulsion (M) and gel (G) bases for diclofenac sodium (DS). The effect of dimethyl sulfoxide (DMSO) as a penetration enhancer was also examined when it was added to the M formulation. To study the in vitro potential of these formulations, permeation studies were performed with Franz diffusion cells using excised dorsal rat skin. To investigate their in vivo performance, a carrageenan-induced rat paw edema model was used. The commercial formulation of DS (C) was used as a reference formulation. The results of the in vitro permeation studies and the paw edema tests were analyzed by repeated-measures analysis of variance. The in vitro permeation studies found that M was superior to G and C and that adding DMSO to M increased the permeation rate. The permeability coefficients (Kp) of DS from M and M+DMSO were higher (Kp=4.9×10⁻³±3.6×10⁻⁴ cm/h and 5.3×10⁻³±1.2×10⁻³ cm/h, respectively) than the Kp of DS from C (Kp=2.7×10⁻³±7.3×10⁻⁴ cm/h) and G (Kp=4.5×10⁻³±4.5×10⁻⁵ cm/h). In the paw edema test, M showed the best permeation and effectiveness, and M+DMSO had nearly the same effect as M. The in vitro and in vivo studies showed that M could be a new, alternative dosage form for effective therapy.     

Body and scale growth of wild Atlantic salmon smolts during seaward emigration

The relationship between scale and body growth for emigrating Atlantic salmon, Salmo salar, smolts was previously not understood and therefore was examined in this study using mark-recapture techniques. The size of smolts at time of recapture was significantly greater than when marked (P = 0.0002). The growth in length of smolts emigrating 5 km over an average of 20 days was 7.7 ± 6.1 mm per day. Instantaneous somatic growth (G _body) ranged from 7.0 × 10⁻⁴ to 5.1 × 10⁻³ (mean = 2.7 × 10⁻³ ± 1.3 × 10⁻³). The mean number of plus growth circuli present per scale was significantly greater for smolts when recaptured compared to when marked (P = 0.0014). The instantaneous growth rate of scales (G _scale) ranged from 1.4 × 10⁻³ to 11.5 × 10⁻³ (mean = 6.6 × 10⁻³ ± 4.3 × 10⁻³). The relationship between body size and scale radius showed positive allometry rather than isometry. The relationship of G _scale with G _body showed positive allometry indicating that scales grew at a slightly faster rate than the body during the emigratory period.     

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

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

A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation

A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat _GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 × 10⁻⁷ and 4.7 × 10⁻⁷ transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H.␣pylori recipients, with pHel2 showing an efficiency of 2.0 × 10⁻⁵ transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a recA-deficient H. pylori mutant by the cloned H. pylorirecA ⁺ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.␣pylori demonstrate the general usefulness of␣this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future. Received: 22 April 1997 / Accepted: 4 November 1997     

Characterisation of acidic sites of Pseudomonas biomass capable of binding protons and cadmium and removal of cadmium via biosorption

Summary The ability of Pseudomonas aeruginosa to accumulate Cd(II) ions from wastewater industries was experimentally investigated and mathematically modelled. From the potentiometric titration and non-ideal competitive analysis (NICA) model, it was found that the biomass contains three acidic sites. The values of proton binding (pK _i =1.66±3.26×10⁻³, 1.92±1.63×10⁻⁴ and 2.16±3.79×10⁻⁴) and binding constant of cadmium metal ions (pK _M1=1.99±2.45×10⁻³ and pK _M2=1.67±4.08×10⁻³) on the whole surface of biomass showed that protonated functional groups and biosorption of Cd(II) ions could be attributed to a monodentate binding to one acidic site, mainly the carboxylic group. From the isothermal sorption experimental data and Langmuir model, it was also found that the value of Langmuir equilibrium (pK _f) constant is 2.04±2.1×10⁻⁵ suggesting that the carboxyl group is the main active binding site. In addition, results showed that the maximum cadmium capacity (q _max) and affinity of biomass towards cadmium metal ions (b) at pH 5.1 and 20 min were 96.5±0.06 mg/g and 3.40×10⁻³± 2.10×10⁻³, respectively. Finally, interfering metal ions such as Pb(II), Cu(II), Cr(III), Zn(II), Fe(II), Mn(II), Ca(II) and Mg(II) inhibited Cd(II) uptake. Comparing the biosorption of Cd(II) by various Pseudomonas isolates from contaminated environment samples (soil and sewage treatment plant) showed that maximum capacities and equilibrium times were different, indicating that there was a discrepancy in the chemical composition between biomasses of different strains.     

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

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

Characterization of Novel Orange Fluorescent Protein Cloned from Cnidarian Tube Anemone <Emphasis Type="Italic">Cerianthus</Emphasis> sp.

A novel orange fluorescent protein (OFP) was cloned from the tentacles of Cnidarian tube anemone Cerianthus sp. It consists of 222 amino acid residues with a calculated molecular mass of 25.1 kDa. A BLAST protein sequence homology search revealed that native OFP has 81% sequence identity to Cerianthus membranaceus green fluorescent protein (cmFP512), 38% identity to Entacmaea quadricolor red fluorescent protein (eqFP611), 37% identity to Discosoma red fluorescent protein (DsRed), 36% identity to Fungia concinna Kusabira-orange fluorescent protein (KO), and a mere 21% identity to green fluorescent protein (GFP). It is most likely that OFP also adopts the 11-strand β-barrel structure of fluorescent proteins. Spectroscopic analysis indicated that it has a wide absorption spectrum peak at 548 nm with two shoulders at 487 and 513 nm. A bright orange fluorescence maximum at 573 nm was observed when OFP was excited at 515 nm or above. When OFP was excited well below 515 nm, a considerable amount of green emission maximum at 513 nm was also observed. It has a fluorescence quantum yield (Φ) of 0.64 at 25°C. The molar absorption coefficients (ɛ) of folded OFP at 278 and 548 nm are 47,000 and 60,000 M-1⁻¹ • cm-1⁻¹, respectively. Its fluorescent brightness (ɛ Φ) at 25°C is 38,400 M⁻¹-1 • cm⁻¹-1. Like other orange-red fluorescent proteins, OFP is also tetrameric. It was readily expressed as soluble protein in Escherichia coli at 37°C, and no aggregate was observed in transfected HeLa cells under our experimental conditions. Fluorescent intensity of OFP is detectable over a pH range of 3 to 12.     

Characterization of a putative pheromone biosynthesis-activating neuropeptide (PBAN) receptor from the pheromone gland of Heliothis peltigera

The binding of [³H]tyrosyl-PBAN28-33NH₂ to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO₃ ⁻ ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a K_d of 5.73 ± 1.05 × 10⁻⁶ M and a Bmax of 1.85 ± 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH₂ and PBAN28-33ΝΗ₂ with a K_i of 4.3 ± 1.1 × 10⁻⁶ M and 4.9 ± 2.6 × 10⁻⁶ M, respectively. Accepted: 4 February 1999     

Anin vitro model for endothelial permeability: Assessment of monolayer integrity

Summary An essential component of anyin vitro model for endothelial permeability is a confluent cell monolayer. The model reported here utilizes primary human umbilical vein endothelial cells (HUVEC) cultured on recently developed polyethylene terephthalate micropore membranes. Using a modification of the Wright-Giemsa stain, confluent HUVEC monolayers grown on micropore membranes were routinely assessed using light microscopy. Determination of confluence using this method was confirmed by scanning electron microscopy. Transendothelial electrical resistance of HUVEC monolayers averaged 27.9±11.4 Ω · cm², 10 to 21% higher than literature values. Studies characterizing the permeability of the endothelial cell monolayer to³H-inulin demonstrated a linear relationship between the luminal concentration of³H-inulin and its flux across HUVEC monolayers. The slope of the flux versus concentration plot, which represents endothelial clearance of³H-inulin, was 2.01±0.076 × 10⁻⁴ ml/min (r²=.9957). The permeability coefficient for the HUVEC monolayer-micropore membrane barrier was 3.17±0.427×10⁻⁶ cm/s with a calculated permeability coefficient of the HUVEC monolayer alone of 4.07±0.617×10⁻⁶ cm/s. The HUVEC monolayer reduced the permeability of the micropore membrane alone to³H-inulin (1.43±0.445×10⁻⁵ cm/s) by 78%. Evans blue dye-labeled bovine serum albumin could not be detected on the abluminal side without disruption of the HUVEC monolayer. These results demonstrate a model for endothelial permeability that can be extensively assessed for monolayer integrity by direct visualization, transendothelial electrical resistance, and the permeability of indicator macromolecules.     

Non-parametric estimation of thresholds for radiation effects in vertebrate species under chronic low-LET exposures

Databases on effects of chronic low-LET radiation exposure were analyzed by non-parametric statistical methods, to estimate the threshold dose rates above which radiation effects can be expected in vertebrate organisms. Data were grouped under three umbrella endpoints: effects on morbidity, reproduction, and life shortening. The data sets were compiled on a simple ‘yes’ or ‘no’ basis. Each data set included dose rates at which effects were reported without further details about the size or peculiarity of the effects. In total, the data sets include 84 values for endpoint “morbidity”, 77 values for reproduction, and 41 values for life shortening. The dose rates in each set were ranked from low to higher values. The threshold TDR5 for radiation effects of a given umbrella type was estimated as a dose rate below which only a small percentage (5%) of data reported statistically significant radiation effects. The statistical treatment of the data sets was performed using non-parametric order statistics, and the bootstrap method. The resulting thresholds estimated by the order statistics are for morbidity effects 8.1 × 10⁻⁴ Gy day⁻¹ (2.0 × 10⁻⁴–1.0 × 10⁻³), reproduction effects 6.0 × 10⁻⁴ Gy day⁻¹ (4.0 × 10⁻⁴–1.5 × 10⁻³), and life shortening 3.0 × 10⁻³ Gy day⁻¹ (1.0 × 10⁻³–6.0 × 10⁻³), respectively. The bootstrap method gave slightly lower values: 2.1 × 10⁻⁴ Gy day⁻¹ (1.4 × 10⁻⁴–3.2 × 10⁻⁴) (morbidity), 4.1 × 10⁻⁴ Gy day⁻¹ (3.0 × 10⁻⁴–5.7 × 10⁻⁴) (reproduction), and 1.1 × 10⁻³ Gy day⁻¹ (7.9 × 10⁻⁴–1.3 × 10⁻³) (life shortening), respectively. The generic threshold dose rate (based on all umbrella types of effects) was estimated at 1.0 × 10⁻³ Gy day⁻¹.     

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4°C with high affinity (K _a = 1.49 ± 0.09 × 10⁹ M⁻¹; 0.17 ± 0.02 × 10⁷ M⁻¹) and low binding capacity (B _max = 3.24 ± 0.84 × 10⁻⁵ M; 0.33 ± 0.06 × 10⁻⁴ M). The binding affinity and capacity of testosterone at 23 and 36°C, respectively were similar to those determined at 4°C. However, oestradiol showed no binding activity at 36°C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36°C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.     

Determination of the kinetics of permeation of dimethyl sulfoxide in isolated corneas

Corneal cryopreservation requires that endothelial cells remain viable and intercellular structure be preserved. High viability levels for cryopreserved endothelial cells have been achieved, but preserving intercellular structure, especially endothelial attachment to Descemet's membrane, has proved difficult. Cell detachment apparently is not caused by ice, suggesting osmotic or chemical mechanisms. Knowledge of the permeation kinetics of cryoprotectants (CPAs) into endothelial cells and stroma is essential for controlling osmotic and chemical activity and achieving adequate tissue permeation prior to cooling. Proton nuclear magnetic resonance (NMR) spectroscopy was used to assess the permeation of dimethyl sulfoxide (DMSO) into isolated rabbit corneas. Corneas with intact epithelia were exposed to isotonic medium or 2.0 mol/L DMSO for 60 min and subsequently transferred to 2.0 or 4.0 mol/L DMSO, respectively, at 22, 0, or −10°C. DMSO concentration in the cornea was measured vs time. The Kedem-Katchalsky model was fitted to the data. Hydraulic permeability (m³/N·s) is 7.1×10⁻¹³+216%-11% at 22°C, 8.2×10⁻¹³+235%−21% at 0°C, and 1.7×10⁻¹⁴+19% −16% at −10°C. The reflection coefficient is 1.0+2%−1% at 22°C and 0°C, and 0.9±5% at −10°C. Solute mobility (cm/s) is 5.9×10⁻⁶+6%–11% at 22°C, 3.1×10⁻⁶+12%−11% at 0°C, and 5.0×10⁻⁸ cm/s+59%−40% at −10°C.     

Placental Alpha Hemoglobin Stabilizing Protein (AHSP) and recurrent miscarriage

AHSP inhibits cellular production of the reactive oxygen species. Reduced AHSP indicates reduced protection against oxidative stressors. Our objective was to investigate AHSP levels in recurrent miscarriage (RM). Trophoblast was collected from women of 10 weeks gestation: voluntary abortion controls (VA, n = 10); spontaneous first miscarriage with subsequent normal pregnancy (SMSN, n = 15) or with subsequent miscarriage (SMSM, n = 5); RM previously investigated (RMPS, n = 5) or not previously investigated (RM, n = 5). AHSP mRNA and protein were determined using real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. One-way ANOVA was performed to assess statistical significance (p < 0.05). ahsp mRNA levels were maximally reduced in RM and RMPS (8.0 × 10⁻⁶ ± 1.3 and 8.1 × 10⁻⁶ ± 0.7, respectively) compared with SMSN and VA (16.1 × 10⁻⁶ ± 2.3 and 26.1 × 10⁻⁶ ± 2.7, respectively). SMSM showed levels significantly reduced as well (9.0 × 10⁻⁶ ± 2.3). In RM, a reduced defense from oxidative stressors is evident at first miscarriage, identifying women at high risk for subsequent eventful pregnancy. Reduced AHSP may identify women at risk of experiencing further miscarriages. Monica Emanuelli and Monia Cecati contributed equally to this paper.