Kinetics of leucrose formation from sucrose by dextransucrase

Leucrose formation from sucrose and fructose by dextransucrase is of practical interest. It has been investigated at different experimental conditions, including the influence of temperature on reaction rate and selectivity. Under appropriate conditions high product yield can be obtained. Furthermore, a model is presented that allows interpretation of the experimental data.     

High-density algal photobioreactors using light-emitting diodes

Lack of high-density algal photobioreactors (PBR) has been a limitation in exploiting the biotechnological potential of algae. Recent developments of highly efficient light-emitting diodes (LED using gallium aluminum arsenide chips) have made the development of a small LED-based PBR possible. We have calculated theoretical values of gas mass transfer requirements and light-intensity requirement to support high-density algal cultures for the 680 nm monochromatic red light from LED as a light source. A prototype PBR has been designed based on these calculations. A cell concentration of more than 2 x 10(9) cells/mL (more than 6.6% v%sol;v), cell doubling times as low as 12 h, and an oxygen production rate as high as 10 mmol oxygen/L culture/h were achieved using on-line ultrafiltration to periodically provide fresh medium. (c) 1994 John Wiley & Sons, Inc.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

Binding of epidermal growth factor and transforming growth factor-alpha in mammalian preimplantation embryos

Preimplantation embryos of the pig (Days 11 to 15), cow (Days 14 to 16), sheep (Day 14) and pony (Day 16) bind epidermal growth factor (EGF) specifically. Binding was not detected in embryos of the rabbit at Day 5 or 6 or the hamster at Day 3. Transforming growth factor-alpha displaced [(125)I] EGF in pig, cow and pony embryos almost as much as unlabeled EGF. The binding affinities of EGF ranged from 12 to 233 pM in pig and cow embryos. The range of species and binding features indicate that the EGF family may play a significant role in mammalian preimplantation development.     

Effect of oxygen concentration in the incubator's gas phase on the development of cultured preimplantation rabbit embryos

Development of rabbit preimplantation embryos cultured under 20, 5 or 1% oxygen was studied. Three-day-old morulae were cultured in a protein-free medium (BSM II supplemented with 5 mg PVP/ml medium) for 24 and 48 h. Embryonic development was evaluated by gross morphology and by incorporation of tritiated thymidine as an indicator of cell proliferation. The lower oxygen concentrations yielded significantly better embryo development at 24 and at 48 h than the 20% concentration. There was no significant difference in development between 5% and 1% oxygen. Addition of the radical scavanger superoxiddismutase (SOD), either alone or in combination with catalase or reduced glutathione, did not improve embryo development even in the 20% oxygen group. Our data suggest the need to reduce in vitro oxygen levels from 20% to more physiological concentrations.     

Influence of photoperiod on the secretion of testosterone as a response to sexual stimulus in male goats

Monthly sexual behavior tests were carried out for 1 yr in a group of 19 male goats (10 Verata breed and 9 Malague?a breed) 12 mo of age. Simultaneously with the tests, 5 blood samples were collected, 2 before and 3 after contact with females, to determine the influence of sexual stimulus on the secretion of testosterone. By means of radioimmunoassay, it was determined that both breeds showed a significant increase in testosterone secretion as a result of the sexual stimulus during increasing daylength (Verata: P<0.01; Malague?a: P<0.05), when testosterone secretion levels prior to the stimulus were the lowest of the year. Moreover, the level of testosterone secretion was higher in the Verata than in the Malague?a breed (P<0.01). A significant though low correlation (P<0.05) between the rise of testosterone levels as a response to sexual stimulus and the degree of sexual behavior in the males was observed in the Verata breed. However, there was no correlation between these factors in Malague?a male goats.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

Effects of NaCl on Flows of N and Mineral Ions and on NO3- Reduction Rate within Whole Plants of Salt-Sensitive Bean and Salt-Tolerant Cotton

The effects of NaCl on the transport rates of cations, NO3-, and reduced N compounds between roots and shoot and on NO3- assimilation rate were examined on plants of two species differing in their sensitivity to salinity, bean (Phaseolus vulgare L. cv Gabriella) and cotton (Gossypium hirsutum L. cv Akala). Biomass production after 20 d in response to 50 and 100 mM NaCl decreased by 48 and 59% in bean, but only 6 and 14% in cotton. The comparison of the flow patterns obtained for control and NaCl-fed plants showed that salinity induced a general decrease in all the fluxes involved in partitioning of N and the various ions. This decrease was markedly higher in bean than in cotton. Within either species, the different flows (uptake, xylem flux, phloem flux) of a given element were affected by NaCl to the same extent with minor exceptions. No specific effect of salinity on any of the components of N partitioning were discerned. The greater sensitivity of nitrate reductase activity to NaCl in bean leaves compared to cotton leaves seems to be due to a decreased compartmentalization of ions rather than to a difference in salt tolerance of the enzyme itself. Overall, our data show that alteration of mineral nutrition is not solely the reflection of a decreased growth rate, but also is a general process that impairs uptake of all the minerals even at mild NaCl salinity.     

Gene technology-based antimetabolite design: the use of an in vitro protein expression system to facilitate antimetabolite design for virally-induced human diseases and malignant conditions

A precondition for the chemotherapeutic treatment of a variety of virally-induced human diseases and malignant conditions is a highly selective interaction of the drug molecule to be used with it's biological target. To ensure the development of novel, effective drugs, it is essential that the biological target is well characterised with regard to it's structure and activity. Such characterisation relies upon adequate amounts of pure target being available. One of the most important enzymatic importers for antimetabolites is the enzyme thymidine kinase. In this article an in vitro protein expression system is described which facilitates the production of milligram amounts of pure and biologically active thymidine kinase, from a number of important biological sources. Results have shown that the in vitro produced enzyme has the exact biochemical propeties of the in vivo enzyme. Thus the in vitro protein expression system is an ideal vechicle to facilitate an in depth investigation of the enzyme's biological properties.