Influence of a Combination of Chemical Enhancers and Iontophoresis on <Emphasis Type="Italic">In Vitro</Emphasis> Transungual Permeation of Nystatin

To promote transungual permeation of nystatin (NYST), molecule with high molecular weight, no water-soluble, amphoteric by iontophoresis. The synergic effect of the combination of cetylpyridinium chloride, CPC, or polyoxyethylene (20) sorbitan monooleate, TW80, and iontophoresis was investigated. In vitro permeation experiments were carried out through bovine hoof slices using vertical diffusion cells. A low current density (0.2 mA/cm²) was applied by introducing Ag/AgCl electrodes in the donor (anode) and receptor (cathode) chambers. The donor phase consisted of a solution, a suspension, or gel-type vehicles containing NYST and surfactants in pH 5.6 HEPES buffer. The addition of CPC to NYST suspension (SOSP) produced a fivefold increase on the permeability of the bovine hoof membrane to the drug. The application of anodal iontophoresis further improved NYST flux. Conversely, NYST transungual permeation was not influenced by TW80 either in the passive diffusion or iontophoretic flux. Furthermore, the iontophoretic treatment does not appear to induce irreversible alterations to the hoof bovine membranes. The present work demonstrated the efficacy of iontophoresis as a treatment for different nail pathologies with large molecules very slightly soluble in water without irreversibly affecting the nail structure. A synergistic effect between CPC and iontophoresis was observed.     

In Vitro Percutaneous Permeation and Skin Accumulation of Finasteride Using Vesicular Ethosomal Carriers

In order to develop a novel transdermal drug delivery system that facilitates the skin permeation of finasteride encapsulated in novel lipid-based vesicular carriers (ethosomes)finasteride ethosomes were constructed and the morphological characteristics were studied by transmission electron microscopy. The particle size, zeta potential and the entrapment capacity of ethosome were also determined. In contrast to liposomes ethosomes were of more condensed vesicular structure and they were found to be oppositely charged. Ethosomes were found to be more efficient delivery carriers with high encapsulation capacities. In vitro percutaneous permeation experiments demonstrated that the permeation of finasteride through human cadaver skin was significantly increased when ethosomes were used. The finasteride transdermal fluxes from ethosomes containing formulation (1.34 ± 0.11 μg/cm²/h) were 7.4, 3.2 and 2.6 times higher than that of finasteride from aqueous solution, conventional liposomes and hydroethanolic solution respectively (P < 0.01).Furthermore, ethosomes produced a significant (P < 0.01) finasteride accumulation in the skin, especially in deeper layers, for instance in dermis it reached to 18.2 ± 1.8 μg/cm². In contrast, the accumulation of finasteride in the dermis was only 2.8 ± 1.3 μg/cm² with liposome formulation. The study demonstrated that ethosomes are promising vesicular carriers for enhancing percutaneous absorption of finasteride.     

Passive and iontophoretic transport enhancement of insulin through porcine epidermis by depilatories: Permeability and fourier transform infrared spectroscopy studies

The effect of thioglycolate-based depilatory lotions was studied on the in vitro passive and iontophoretic permeability of insulin through porcine epidermis and biophysical changes in the stratum corneum (SC) lipids and proteins. The porcine epidermis and Franz diffusion cells modified for iontophoresis were used for the in vitro transport studies. Cathodal iontophoresis was performed at 0.2 mA/cm² current density. Resistance of the control- and depilatory-lotion-treated epidermis was determined according to Ohmslaw. Biophysical changes were studied on porcine SC before (control) and after treatment with the depilatory lotions using Fourier transform infrared (FT-IR) spectroscopy. Asymmetric (∼2915 cm⁻¹) and symmetric (∼2848 cm⁻¹) Carbon-Hydrogen (C-H) stretching absorbances were studied to estimate the extent of lipid extraction. Fourier self-deconvolution and second derivative procedures were applied to amide I band (1700–1600 cm⁻¹) in order to estimate quantitatively the changes in the secondary structure of the SC protein. The passive permeability of insulin was significantly (P<.05) increased through depilatory-lotion-treated (ie, Better Off, Marzena, and Sally Hansen) epidermis in comparison to control. Iontophoresis significantly enhanced (P<.05) the permeability of insulin through depilatory-pretreated epidermis in comparison with the control epidermis. Further, we were able to achieve the desired flux of insulin (5.25 U/cm²/d) through Better Off-treated epidermis using 0.2 mA/cm² current density and 100 U/mL donor concentration of insulin. The SC treated with depilatory lotions showed a decrease in peak areas of C-H stretching absorbances in comparison with untreated SC. Depilatory lotion treatment also decreased (P<.05) the epidermal resistance in comparison with the control epidermis. The decrease in the α-helix conformation and the increase in the random and turn structures were observed in the SC proteins due to depilatory lotion treatment. The changes in the secondary structure of proteins and lipid extraction from the SC are suggested as the cause of the decrease in the epidermal resistance and the increase in the passive and iontophoretic permeability of insulin through depilatory-pretreated epidermis in comparison with the control epidermis.     

Drug-Cyclodextrin-Vesicles Dual Carrier Approach for Skin Targeting of Anti-acne Agent

In the present study attempt was made for preparation of isotretinoin-hydroxypropyl β cyclodextrin (HP-β-CD) inclusion complex and encapsulate this complex in elastic liposomes to study the effect of dual carrier approach on skin targeting of isotretinoin. The isotretinoin HP-β-CD complex was prepared by freeze-drying method and characterized by IR spectroscopy. The drug and drug-CD complex loaded elastic liposomal formulation were prepared and characterized in vitro, ex-vivo and in vivo for shape, size, entrapment efficiency, no. of vesicles per cubic mm, in vitro skin permeation and deposition study, photodegradation and skin toxicity assay. The transdermal flux for different vesicular formulations was observed between 10.5 ± 0.5 to 13.9 ± 1.6 μg/cm²/h. This is about 15-21 folds higher than that obtained from drug solution (0.7 ± 0.1 μg/cm²/h) and 4-5 folds higher than obtained with drug-CD complex solution (2.7 ± 0.1 μg/cm²/h). The amount of drug deposit was found to increase significantly (p < 0.05) by cyclodextrin complexation (30.1 ± 0.1 μg). The encapsulation of this complex in elastic liposomal formulation further increases its skin deposition (262.2 ± 21 μg). The results of skin irritation study using Draize test also showed the significant reduction in skin irritation potential of isotretinoin elastic liposomal formulation in comparison to free drug. The results of the present study demonstrated that isotretinoin elastic liposomal formulation possesses great potential for skin targeting, prolonging drug release, reduction of photodegradation, reducing skin irritation and improving topical delivery of isotretinoin.     

Cavamax W7 composite ethosomal gel of clotrimazole for improved topical delivery: development and comparison with ethosomal gel

The present research work was aimed to formulate clotrimazole encapsulated Cavamax W7 composite ethosomes by injection method for improved delivery across epidermis. 3² factorial design was used to design nine formulations (F1-F9) and compared with ethosomal formulations (F10-F12). F9 with vesicle size of 202.8 ± 4.8 nm, highest zeta potential (−83.6 ± 0.96 mV) and %EE of 98.42 ± 0.15 was selected as optimized composite ethosome and F12 as reference ethosomal formulation. As revealed by transmission electron microscopy F9 vesicles were more condensed, uniformly spherical in shape than F12 vesicles. Vesicular stability studies indicated F9 to be more stable as compared to F12. Both F9 and F12 were incorporated in carbopol 934 gel base to get G1–G8 gel formulations and evaluated for in vitro skin permeability. Cavamax W7 composite ethosomal optimized gel (G5) showed higher in vitro percent cumulative drug permeation (88.53 ± 2.10%) in 8 h and steady state flux (J _ss) of 3.39 ± 1.45 μg/cm²/min against the J _ss of 1.57 ± 0.23 μg/cm²/min for ethosomal gel (G1) and 1.13 ± 0.06 μg/cm²/min for marketed formulation. The J _ss flux of G5 was independent of amount of drug applied/unit area of skin. In vivo confocal laser scanning microscopic study of G5 depicted uniform and deeper penetration of rhodamine B (marker) in epidermis from Cavamax W7 composite ethosomal gel in comparison to G1. Finally, G5 demonstrated better (p < 0.05) antifungal activity against Candida albicans and Aspergillus niger than G1 thus, signifying that Cavamax W7 composite ethosomes present a superior stable and efficacious vesicular system than ethosomal formulation for topical delivery of clotrimazole.     

Permeation and Distribution of Ferulic Acid and Its α-Cyclodextrin Complex from Different Formulations in Hairless Rat Skin

Ferulic acid (FA) is a natural product that occurs in seeds of many plants where it is generally located in the bran. This compound is a multifunctional ingredient endowed with antioxidative, radical scavenging, sunscreening and antibacterial actions. The aim of this study was to analyse the ferulic acid cutaneous permeation and distribution, through and into the skin layers, from different cosmetic vehicles, an O/W emulsion (pH 6.0) and two gel-type formulations at different pH levels (6.0 and 7.4), containing FA alone or an inclusion complex with α-cyclodextrin (CD-FA). In vitro permeation studies were performed in vertical diffusion cells using hairless rat excised skin. At appropriate intervals of time, the amount of permeated sunscreen/radical scavenger was evaluated by high-performance liquid chromatography (HPLC). At the end of experiments, treated skin samples were sectioned with a cryomicrotome and the FA content of the individual slices was analysed by HPLC. FA-containing formulations, O/W emulsion, gels A and B, originated FA fluxes of 8.48 ± 2.31, 8.38 ± 0.89 and 5.72 ± 0.50 μg/cm² h, respectively, thus suggesting the pH influence on FA percutaneous permeation. The use of the inclusion complex, CD-FA, determined in all cases a decrease of FA transdermal permeation while no influence of pH was observed. Gel-type formulations containing FA ensured higher sunscreen storage in the superficial layers if compared with O/W emulsion. When FA was included in α-cyclodextrin, FA amount retained into skin layers decreased markedly.     

Content of the <Emphasis Type="Italic">Alternaria</Emphasis> mycotoxin tenuazonic acid in food commodities determined by a stable isotope dilution assay

The Alternaria mycotoxin tenuazonic acid (TA) was quantified in fruit juices (n = 50), cereals (n = 12) and spices (n = 38) using a recently developed stable isotope dilution assay (SIDA). [¹³ C₆,¹⁵ N]-TA was used as the internal standard. Method validation revealed low limits of detection (LODs) of 0.15 μg/kg (fruit juices), 1.0 μg/kg (cereals) and 17 μg/kg (spices). The respective limits of quantitation were about three times higher. Recovery was about 100% for all matrices. The precision (relative standard deviation of replicate analyses of naturally contaminated samples) was 4.2% (grape juice; 1.7 μg/kg), 3.5% (whole wheat flour; 36 μg/kg) and 0.9% (curry powder; 215 μg/kg). The median content of TA in the analyzed samples was 1.8 μg/kg (fruit juices), 16 μg/kg (cereals) and 500 μg/kg (spices). Positive samples amounted to 86% (fruit juices), 92% (cereals) and 87% (spices).     

Investigation of the Physicochemical and Physicomechanical Properties of a Novel Intravaginal Bioadhesive Polymeric Device in the Pig Model

The purpose of this study was to develop and evaluate the bioadhesivity, in vitro drug release, and permeation of an intravaginal bioadhesive polymeric device (IBPD) loaded with 3′-azido-3′-deoxythymidine (AZT) and polystyrene sulfonate (PSS). Modified polyamide 6,10, poly(lactic-coglycolic acid), polyacrylic acid, polyvinyl alcohol, and ethylcellulose were blended with model drugs AZT and PSS as well as radio-opaque barium sulfate (BaSO4) and then compressed into caplet devices on a tableting press. One set of devices was coated with 2% w/v pentaerythritol polyacrylic acid (APE-PAA) while another remained uncoated. Thermal analysis was performed on the constituent polymers as well the IBPD. The changes in micro-environmental pH within the simulated human vaginal fluid due to the presence of the IBPD were assessed over a period of 30 days. Textural profile analysis indicated that the bioadhesivity of the APE-PAA-coated devices (3.699 ± 0.464 N; 0.0098 ± 0.0004 J) was higher than that of the uncoated devices (1.198 ± 0.150 N; 0.0019 ± 0.0001 J). In addition, BaSO4-facilitated X-ray imaging revealed that the IBPD adhered to pig vaginal tissue over the experimental period of 30 days. Controlled drug release kinetics was obtained over 72 days. During a 24-h permeation study, an increase in drug flux for both AZT (0.84 mg cm⁻² h⁻¹) and PSS (0.72 mg cm⁻² h⁻¹) was realized up to 12 h and thereafter a steady-state was achieved. The diffusion and dissolution dynamics were mechanistically deduced based on a chemometric and molecular structure modeling approach. Overall, results suggested that the IBPD may be sufficiently bioadhesive with desirable physicochemical and physicomechanical stability for use as a prolonged intravaginal drug delivery device.     

Zinc,Manganese, Calcium,Copper, and Cadmium Level in Scalp Hair Samples of Schizophrenic Patients

The purpose of the study was to determine the concentration of trace elements present in scalp hair sample of schizophrenic patients and to find out the relationship between trace elements level and nutritional status or socioeconomic factors. The study was conducted among 30 schizophrenic male patients and 30 healthy male volunteers. Patients were recruited from Bangabandhu Sheikh Mujib Medical University by random sampling. Hair trace element concentrations were determined by flame atomic absorption spectroscopy and analyzed by independent t test, Pearson’s correlation analysis, regression analysis, and analysis of variance (ANOVA). Mn, Zn, Ca, Cu, and Cd concentrations of schizophrenic patients were 3.8 ± 2.31 μg/gm, 171.6 ± 59.04 μg/gm, 396.23 ± 157.83 μg/gm, 15.40 ± 5.68 μg/gm, and 1.14 ± 0.89 μg/gm of hair sample, while those of control subjects were 4.4 ± 2.32 μg/gm, 199.16 ± 27.85 μg/gm, 620.9 ± 181.55 μg/gm, 12.23 ± 4.56 μg/gm, and 0.47 ± 0.32 μg/gm of hair sample, respectively. The hair concentration of Zn and Ca decreased significantly (p = 0.024; p = 0.000, respectively) and the concentration of Cu and Cd increased significantly (p = 0.021; p = 0.000, respectively) in schizophrenic patients while the concentration of Mn (p = 0.321) remain unchanged. Socioeconomic data reveals that most of the patients were poor, middle-aged and divorced. Mean body mass indices (BMIs) of the control group (22.26 ± 1.91 kg/m²) and the patient group (20.42 ± 3.16 kg/m²) were within the normal range (18.5−25.0 kg/m²). Pearson’s correlation analysis suggested that only Ca concentration of patients had a significant positive correlation with the BMI (r = 0.597; p = 0.000) which was further justified from the regression analysis (R ² = 44%; t = 3.59; p = 0.002) and one-way ANOVA test (F = 3.62; p = 0.015). A significant decrease in the hair concentration of Zn and Ca as well as a significant increase in the hair concentration of Cu and Cd in schizophrenic patients than that of its control group was observed which may provide prognostic tool for the diagnosis and treatment of this disease. However, further work with larger population is suggested to examine the exact correlation between trace element level and the degree of disorder.     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.     

The viability to a wall shear stress and propagation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> in the intensive membrane bioreactor

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Kinetic analysis of boron transport in Chara

The permeability of biological membranes to boric acid was investigated using the giant internodal cells of the charophyte alga Chara corallina (Klein ex Will. Esk. R.D. Wood). The advantage of this system is that it is possible to distinguish between membrane transport of boron (B) and complexing of B by plant cell walls. Influx of B was found to be rapid, with equilibrium between the intracellular and extracellular phases being established after approximately 24 h when the external concentration was 50 μM. The intracellular concentration at equilibrium was 55 μM, which is consistent with passive distribution of B across the membrane along with a small amount of internal complexation. Efflux of B occurred with a similar half-time to influx, approximately 3 h, which indicates that the intracellular B was not tightly complexed. The concentration dependence of short-term influx measured with ¹⁰B-enriched boric acid was biphasic. This was tentatively attributed to the operation of two separate transport systems, a facilitated system that saturates at 5 μM, and a linear component due to simple diffusion of B through the membrane. V _max and K _m for the facilitated transport system were 135 pmol m⁻²s⁻¹ and 2 μM, respectively. The permeability coefficient for boric acid in the Chara plasmalemma estimated from the slope of the linear influx component was 4.4 × 10⁻⁷cm s⁻¹ which is an order of magnitude lower than computed from the ether:water partition coefficient for B. Received: 14 August 2000 / Accepted: 16 September 2000     

Transdermal Delivery of an Anti-Cancer Drug via W/O Emulsions Based on Alkyl Polyglycosides and Lecithin: Design,Characterization, and <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of the Possible Irritation Potential in Rats

The purpose of this work was to develop w/o emulsions that could be safely used to promote transdermal delivery of 5-fluorouracil (5-FU). Two pseudo-ternary phase diagrams comprising oleoyl-macrogol glycerides, water, and a surfactant/co-surfactant (S/CoS) mixture of lecithin, ethanol, and either coco glucoside or decyl glucoside were investigated for their potential to develop promising 5-FU emulsions. Six systems were selected and subjected to thermodynamic stability tests; heat–cool cycles, centrifugation, and finally freeze–thaw cycles. All systems passed the challenges and were characterized for transmission electron microscopy, droplet size, rheological behavior, pH, and transdermal permeation through newly born mice skin in Franz diffusion cells. The systems had spherical droplets ranging in diameter from 1.81 to 2.97 μm, pH values ranging from 7.50 to 8.49 and possessed Newtonian flow. A significant (P < 0.05) increase in 5-FU permeability parameters as steady-state flux, permeability coefficient was achieved with formula B5 comprising water (5% w/w), S/CoS mixture of lecithin/ethanol/decyl glucoside (14.67:12.15:18.18% w/w, respectively) and oleoyl-macrogol glycerides (50% w/w). When applied to shaved rat skin, this system was well tolerated with only moderate skin irritation that was recovered within 12 h. Indeed, minor histopathologic changes were observed after 5-day treatment. Further studies should be carried out, in the future, to investigate the potentiality of this promising system to promote transdermal delivery of 5-FU through human skin.     

Cultivation of the green alga, Codium fragile (Suringar) Hariot, by artificial seed production in Korea

Codium fragile (Suringar) Hariot is an edible green alga farmed in Korea using seed stock produced from regeneration of isolated utricles and medullary filaments. Experiments were conducted to reveal the optimal conditions for nursery culture and out-growing of C. fragile. Sampling and measurement of underwater irradiance were carried out at farms cultivating C. fragile at Wando, on the southwestern coast of Korea, from October 2004 to August 2005. Growth of erect thalli and underwater irradiance were measured over a range of depths for three culture stages. During the nursery cultivation stage (Stage I), growth rate was greatest at 0.5 m depth (0.055 ± 0.032 mm day⁻¹), where the average midday irradiance over 60 days was 924 ± 32 μmol photons m⁻² s⁻¹. During the pre-main cultivation stage (Stage II), the greatest growth rate occurred at a depth of 2 m (0.113 ± 0.003 mm day⁻¹) with an average irradiance of 248 ± 116 μmol photons m⁻² s⁻¹. For the main cultivation stage (Stage III) of the alga, thalli achieved the greatest increase in biomass at 1 m depth (7.2 ± 1.0 kg fresh wt m⁻¹). These results suggest that optimal growth at each cultivation stages of C. fragile could be controlled by depth of cultivation rope.     

Expansion of a direct shoot organogenesis system in peanut (Arachis hypogaea L.) to include US cultivars

Agrobacterium-mediated transformation, employing direct shoot organogenesis, allows for mature transgenic plants to be obtained quickly (3–4 mo). In this study, peanut (Arachis hypogaea L.) cultivars Florida-07, Georgia Green, Georgia Brown, New Mexico Valencia A, and VC-2 were selected to test their shoot induction response for use in future transformation experiments. Two types of cotyledon explants were examined, those that previously had an attached embryo axis upon cotyledon separation (explant A) and those that were embryo axis-free upon separation (explant B). Explants were placed onto a shoot induction medium with N ⁶-benzyladenine concentrations ranging from 10–80 μM for Florida-07, Georgia Green, and VC-2; 10–20 μM for Georgia Brown; and 10–640 μM for New Mexico Valencia A. Following a 4-wk culture period, explants were visually rated based on a scale of 1–4, where 1 indicated slight greening, but no growth, and 4 indicated greening, adventitious bud formation, as well as small leaf expansion. A difference in shoot induction was observed for the cotyledon explants examined (P > t = <0.0001). Explant A had greater shoot induction with a visual rating of 1.8 ± 0.1; explant B had a rating of 1.6 ± 0.1 (P > t = <0.0001). Additionally, cultivars responded to the culture conditions differently (cultivar × N ⁶-benzyladenine interaction). Georgia Green on 10 μM N ⁶-benzyladenine produced the most shoot buds (24.6%) and the highest visual rating (2.1), followed by VC-2 on 10 μM N ⁶-benzyladenine (22.1%, 1.8), New Mexico Valencia A on 640 μM N ⁶-benzyladenine (21.4%, 1.8), Georgia Brown on 80 μM N ⁶-benzyladenine (9.0%, 1.7), and Florida-07 on 40 μM N ⁶-benzyladenine (7.1%, 1.8). Of the tested varieties, Georgia Green, New Mexico Valencia A, and VC-2 were best suited for future transformation experiments based on their shoot bud production.     

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 × 10⁻² U mg⁻¹, respectively. The Michaelis–Menten constant (K _m values) and the maximal reaction velocities (V _max values) of CelEdx16 were 47.1 μM and 9.6 × 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 × 10⁻⁵ μmole min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40°C.     

Expression of the Zinc Transporters Genes and Metallothionein in Obese Women

Research has investigated the participation of zinc transport proteins and metallothionein in the metabolism of this mineral. However, studies about the genetic expression of these proteins in obese patients are scarce. The study determined the expression of zinc transporter protein codifying genes (ZnT-1, Zip-1 and Zip-3) and of metallothionein in 55 obese women, aged between 20 and 56 years. The assessment of body composition was carried out using anthropometric measurements and bioelectrical impedance. Zinc intake was obtained by recording diet over a 3-day period, and the nutritional analysis was carried out using NutWin software version 1.5. The plasmatic and erythrocytary zinc were analyzed by atomic absorption spectrophotometry (λ = 213. 9 nm). The determination of mRNA expression of the zinc transporter proteins and metallothionein was carried out using blood, using the RT-PCR method. The mean values of body mass index were 37.9 ± 5.5 kg/m². The average intake of zinc was 9.4 ± 2.3 mg/day. The analysis of the zinc plasma concentrations showed values of 58.4 ± 10.9 μg/dL. The mean values of zinc in the erythroytes were 38.7 ± 9.1 μg/g Hb. The metallothionein gene had a higher expression in the blood, when compared to zinc transporters ZnT-1, Zip-1, and Zip-3 (p = 0.01). The study shows that there are alterations in the biochemical parameters of zinc in obese patients assessed, as well as higher expression of the codifying gene metallothionein, when compared to the investigated zinc transporters.     

Plasma Leptin,Plasma Zinc,and Plasma Copper Are Associated in Elite Female and Male Judo Athletes

The purpose of this study was to compare plasma leptin, plasma zinc, and plasma copper levels and their relationship in trained female and male judo athletes (n = 10 women; n = 8 men). Blood samples were obtained 24 h after training to measure plasma zinc, copper, and leptin levels. Subjects presented similar values to age (22 ± 2 years old), body mass index (24 ± 1 kg/m²), plasma zinc (17.2 ± 2 μmol/L), copper (12.5 ± 2 μmol/L), and leptin (5.6 ± 1.3 μg/L). However, height, total body mass, lean mass, fat mass, and sum of ten-skinfold thickness were higher in male than female. Plasma leptin was associated with sum of ten skinfolds in male (r = 0.91; p < 0.001) and female athletes (r = 0.84; p < 0.003). Plasma zinc was associated with leptin in males (r = 0.82; p < 0.05) while copper was associated with plasma leptin in females (r = 0.66; p < 0.05). Our results suggest that young judo athletes lost sex-related differences in leptin levels. Plasma zinc, plasma copper, and energy homeostasis may be involved in regulation of plasma leptin.     

Decrease of phosphoribulokinase activity by antisense RNA in transgenic tobacco: definition of the light environment under which phosphoribulokinase is not in large excess

 To test the hypothesis that the contribution of phosphoribulokinase (PRK) to the control of photosynthesis changes depending on the light environment of the plant, the response of transgenic tobacco (Nicotiana tabacum L.) transformed with antisense PRK constructs to irradiance was determined. In plants grown under low irradiance (330 μmol m⁻² s⁻¹) steady-state photosynthesis was limited in plants with decreased PRK activity upon exposure to higher irradiance, with a control coefficient of PRK for CO₂ assimilation of 0.25 at and above 800 μmol m⁻² s⁻¹. The flux control coefficient of PRK for steady-state CO₂ assimilation was zero, however, at all irradiances in plant material grown at 800 μmol m⁻² s⁻¹ and in plants grown in a glasshouse during mid-summer (alternating shade and sun 300–1600 μmol m⁻² s⁻¹). To explain these differences between plants grown under low and high irradiances, Calvin cycle enzyme activities and metabolite content were determined. Activities of PRK and other non-equilibrium Calvin cycle enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase-oxygenase were twofold higher in plants grown at 800 μmol m⁻² s⁻¹ or in the glasshouse than in plants grown at 330 μmol m⁻² s⁻¹. Activities of equilibrium enzymes transketolase, aldolase, ribulose-5-phosphate epimerase and isomerase were very similar under all growth irradiances. The flux control coefficient of 0.25 in plants grown at 330 μmol m⁻² s⁻¹ can be explained because low ribulose-5-phosphate content in combination with low PRK activity limits the synthesis of ribulose-1,5-bisphosphate. This limitation is overcome in high-light-grown plants because of the large relative increase in activities of sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase under these conditions, which facilitates the synthesis of larger amounts of ribulose-5-phosphate. This potential limitation will have maintained evolutionary selection pressure for high concentrations of PRK within the chloroplast. Received: 15 November 1999 / Accepted: 27 January 2000