Seasonal abundance and distribution of <Emphasis Type="Italic">Vibrio</Emphasis> species in the treated effluent of wastewater treatment facilities in suburban and urban communities of Eastern Cape Province,South Africa

We assessed the seasonal abundance and distribution of Vibrio species as well as some selected environmental parameters in the treated effluents of two wastewater treatment plants (WWTP), one each located in a suburban and urban community of Eastern Cape Province, South Africa. Vibrio population density ranged from 2.1×10⁵ to 4.36×10⁴ CFU/ml in the suburban community and from 2.80×10⁵ to 1.80×10⁵ CFU/ml in the urban community. Vibrio species associated with 180 μ, 60 μ, and 20 μ plankton sizes were observed at densities of 0–136×10³ CFU/ml, 0–8.40×10² CFU/ml, and 0–6.80×10² CFU/ml, respectively at the suburban community’s WWTP. In the urban community, observed densities of culturable Vibrio were 0–2.80×10² CFU/ml (180 μ), 0–6.60×10² CFU/ml (60 μm), and 0–1.80× 10³ CFU/ml (20 μm). The abundance of free-living Vibrio species ranged from 0 to 1.0×10² and 1.0×10³ CFU/ml in the suburban and urban communities’ WWTPs, respectively. Molecular confirmation of the presumptive Vibrio isolates revealed the presence of V. fluvialis (41.38%), V. vulnificus (34.48%), and V. parahaemolyticus (24.14%) in the suburban community effluents. In the urban community molecular confirmation revealed that the same species were present at slightly different percentages, V. fluvialis (40%), V. vulnificus (36%), and V. parahaemolyticus (24%). There was no significant correlation between Vibrio abundance and season, either as free-living or plankton-associated entities, but Vibrio species abundance was positively correlated with temperature (r=0.565; p<0.01), salinity, and dissolved oxygen (p<0.05). Turbidity and pH showed significant seasonal variation (p<0.05) across the seasons in both locations. This study underscores the potential of WWTPs to be sources of Vibrio pathogens in the watershed of suburban and urban communities in South Africa.     

Accelerated Biodegradation of High and Low Concentrations of p-Nitrophenol (PNP) by Bacterial Inoculation in Industrial Wastewater: The Role of Inoculum Size on Acclimation Period

The effect of inoculum size on the acclimation period and rate and extent of p-nitrophenol (PNP) degradation at high (1–10 mg/L) and low (26 μg/L) concentrations for two bacteria was determined in defined media as well as industrial wastewater. Increased inoculum size did not affect the acclimation period of either bacterium at high (1–10 mg/L) PNP concentrations. At low PNP concentrations (26 μg/L), the two bacteria behaved differently. The acclimation period was shortened and both the rate and extent of mineralization of PNP were enhanced by increasing the Corynebacterium sp. inoculum size from 3 × 10⁵ to 3 × 10⁶ cells/ml. Addition of phosphate or elimination of predators also reduced the acclimation period. Conversely, increasing the inoculum size from 3 × 10⁵ to 5 × 10⁶ cells/ml of Pseudomonas putida lengthened the acclimation period and reduced both the rate and extent of mineralization. It is suggested that, in a given environment, the success of an introduced species to enhance the degradation of a chemical depends upon (i) concentration of the chemical, (ii) selection of an appropriate microorganism, and (iii) utilization of a suitable inoculum size. Received: 1 April 1996 / Accepted: 6 May 1996     

Growth and net photosynthetic rates of <Emphasis Type="Italic">Hosta</Emphasis> ‘blue vision’ during acclimatization in bright,natural light with CO<Subscript>2</Subscript> enrichment

Summary Hosta ‘Blue Vision’, a shade-adapted perennial, was successfully acclimatized in high, natural light conditions in the research Acclimatron^TM at Clemson University, Clemson, SC during the summer of 2000. The supplemental CO₂ levels achieved during acclimatization were 710±113, 2396±121, and 5641±119 μmol mol⁻¹, approximately 2×, 6×, and 15× ambient CO₂. Plants were maintained in H₂O-saturated atmospheres and protected from temperature increases associated with high light intensity. In the 5 wk following ex vitro transfer, plantlet roots grew at the 2× CO₂ level, but shoot biomass was unaffected. Results for the 6× and 15× CO₂ levels were comparable and provided the best plantlet growth. The “doubling time’ that is characteristic of exponential growth was 10.8 and 9.8 d for root and shoot dry weights, respectively. There was no indication of light saturation of net photosynthetic rate (NPR) over the photosynthetic photon flux density (PPFD) range of 100–1200 μmolm⁻²s⁻¹ experienced during this study. An interaction between CO₂ and light intensity levels was detected for NPR of Hosta ‘Blue Vision’ with CO₂ saturation occurring at approximately 2800 μmol mol⁻¹. regardless of light level. Furthermore, at the optimal CO₂ level, NPR increased quadratically as light intensity increased, and NPR was greatest at the maximum light intensity (PPFD: 1200 μmol m⁻²s⁻¹).     

Increased PC-1 phosphodiesterase activity and inhibition of glucose uptake in adipocytes of type 2 diabetic rats

This study was designed to understand the cellular mechanisms responsible for defects in the insulin-stimulated signal transduction pathway in a type 2 diabetic animal model. We examined the in vitro PC-1 phosphodiesterase activity and glucose uptake in adipose tissue of streptozotocin (STZ)-induced type 2 diabetic rats. The PC-1 activity was significantly increased in adipose tissue of diabetic rats (0.54 ± 0.08 nmol PNTP hydrolyzed/mg protein/min) compared with controls (0.29 ± 0.05 nmol PNTP hydrolyzed/mg protein/min, p < 0.05). Upon insulin stimulation (100 nM), glucose uptake in the adipose tissue of the controls (4.17 ± 1.28×10⁻⁸ μmol/mg/min) was significantly higher than that in the diabetic rats (1.26 ± 0.35×10⁻⁸; p < 0.05). These results suggest that elevated PC-1 phosphodiesterase activity and decreased glucose uptake in adipose tissues may be acquired characteristics contributing to the development of type 2 diabetes mellitus.     

Cell growth and nutritive value of the tropical benthic diatom, <Emphasis Type="Italic">Amphora</Emphasis> sp., at varying levels of nutrients and light intensity,and different culture locations

Two series of experiments were conducted to determine suitable growth factors for the mass propagation of the local algal isolate Amphora sp. A higher growth rate of 0.2 doubling (μ) day⁻¹ was attained at a lower photosynthetic photon flux density (PPFD; 11.4 μmol photon m⁻²s⁻¹) compared to cultures exposed to higher levels of PPFD (16.1 μmol photon m⁻²s⁻¹, −0.1 μ day ⁻¹; 31.3 μmol photon m⁻²s⁻¹, 0.0 μ day⁻¹). Cultures located inside the laboratory had a significantly higher cell density (133 × 10⁴ cells cm⁻²) and growth rate (0.3 μ day⁻¹) compared to those located outdoors (100 × 10⁴ cells cm⁻², 0.2 μ day⁻¹). A comparison of nutrient medium across two locations showed that lipid content was significantly higher in cultures enriched with F/2MTM (macronutrients + trace metals) and F/2MV (macronutrients + vitamins). Saturated fatty acids were also present in high concentrations in cultures enriched with F/2M (macronutrients only). Significantly higher amounts of saturated fatty acids were observed in cultures located outdoors (33.1%) compared to those located indoors (26.6%). The protein, carbohydrates and n-6 fatty acid content of Amphora sp. were influenced by the location and enrichment of the cultures. This study has identified growth conditions for mass culture of Amphora sp. and determined biochemical composition under those culture conditions. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha

Nitrosomonas eutropha, an obligately lithoautotrophic bacterium, was able to nitrify and denitrify simultaneously under anoxic conditions when gaseous nitrogen dioxide (NO₂) was supplemented to the atmosphere. In the presence of gaseous NO₂, ammonia was oxidized, nitrite and nitric oxide (NO) were formed, and hydroxylamine occurred as an intermediate. Between 40 and 60% of the produced nitrite was denitrified to dinitrogen (N₂). Nitrous oxide (N₂O) was shown to be an intermediate of denitrification. Under an N₂ atmosphere supplemented with 25 ppm NO₂ and 300 ppm CO₂, the amount of cell protein increased by 0.87 mg protein per mmol ammonia oxidized, and the cell number of N. eutropha increased by 5.8 × 10⁹ cells per mmol ammonia oxidized. In addition, the ATP and NADH content increased by 4.3 μmol ATP (g protein)^–1 and 6.3 μmol NADH (g protein)^–1 and was about the same in both anaerobically and aerobically grown cells. Without NO₂, the ATP content decreased by 0.7 μmol (g protein)^–1, and the NADH content decreased by 1.2 μmol (g protein)^–1. NO was shown to inhibit anaerobic ammonia oxidation. Received: 9 October 1996 / Accepted: 5 December 1996     

Direct rooting and hardening of tea microshoots in the field

Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) has been widely attempted but commercial exploitation of this method is limited by heavy losses during the hardening procedures. In the present study, optimization of time of harvesting (spring and early summer) of microshoots, shoot size, soil pH (4.0–6.4), plant growth regulator treatment (IBA; 500 mg l^-1, 30 min) CO₂ (9.09/10×10⁻⁵ mol l^-1 to 10.22/10×10^-5 mol l^-1 and 20/11×10⁻⁵ mol l^-1 to 80/13×10⁻⁷ mol l^-1) enrichment and light (15 μ mol m^-2 s^-1) conditions in specially designed hardening chambers, made a significant impact on the percent of success for hardening. Following the standardized procedure, up to 71.6% root induction and 73% survival could be achieved. Successful field transfer was also accomplished. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Physiological differences in the growth of <Emphasis Type="Italic">Sargassum horneri</Emphasis> between the germling and adult stages

The effects of temperature, irradiance, and daylength on Sargassum horneri growth were examined at the germling and adult stages to discern their physiological differences. Temperature–irradiance (10, 15, 20, 25, 30°C × 20, 40, 80 μmol photons m⁻²s⁻¹) and daylength (8, 12, 16, 24 h) experiments were carried out. The germlings and blades of S. horneri grew over a wide range of temperatures (10–25°C), irradiances (20–80 μmol photons m⁻²s⁻¹), and daylengths (8–24 h). At the optimal growth conditions, the relative growth rates (RGR) of the germlings were 21% day⁻¹ (25°C, 20 μmol photons m⁻²s⁻¹) and 13% day⁻¹ (8 h daylength). In contrast, the RGRs of the blade weights were 4% day⁻¹ (15°C, 20 μmol photons m⁻²s⁻¹) and 5% day⁻¹ (12 h daylength). Negative growth rates were found at 20 μmol photons m⁻²s⁻¹ of 20°C and 25°C treatments after 12 days. This phenomenon coincides with the necrosis of S. horneri blades in field populations. In conclusion, we found physiological differences between S. horneri germlings and adults with respect to daylength and temperature optima. The growth of S. horneri germlings could be enhanced at 25°C, 20 μmol photons m⁻²s⁻¹, and 8 h daylength for construction of Sargassum beds and restoration of barren areas.     

Copper modifies the activity of sodium-transporting systems in erythrocyte membrane in patients with essential hypertension

The aim of the study was to verify the hypothesis if copper could influence the activity of sodium-transporting systems in erythrocyte membrane that could be related to essential hypertension. The examined group of patients consisted of 15 men with hypertension. The control group was 11 healthy male volunteers. The Na⁺/H⁺ exchanger (NHE) activity in erythrocytes was determined according to Orlov et al. The activity of transporting systems (ATP-Na⁺/K⁺; co-Na⁺/K⁺/Cl⁻; ex-Na⁺/Li⁺; free Na⁺ and K⁺ outflow [Na⁺, K⁺-outflow]) was determined according to Garay's method. The concentration of copper in plasma was assessed using atomic absorption spectrometry. The activity of ATP-Na⁺/K⁺ (μmol/L red blood cells [RBCs]/h) in hypertensive patients was 2231.5±657.6 vs 1750.5±291 in the control (p<0.05), the activity of co-Na⁺/K⁺/Cl⁻ (μmol/L RBCs/h) in hypertensives was 171.3±77.9 vs 150.7±53.9 in the control (NS). Na⁺-outflow (μmol/L RBCs/h) in hypertensives was 118.3±51.6 vs 113.3±24.4 in the control (NS). The K⁺-outflow (μmol/L RBCs/h) in hypertensives was 1361.7±545.4 vs 1035.6±188.3 in the control (NS). The activity of ex-Na⁺/Li⁺ (μmol/L RBCs/h) in hypertensive patients was 266.1±76.1 vs 204.1±71.6 in the control (p<0.05). NHE activity (mmol/L RBCs/h) in hypertensives was 9.7±2.96 vs 7.7±1.33 in the control (p<0.05). In hypertensive patients, negative correlation was found between the activity of Na⁺/K⁺/Cl⁻ co-transport and plasma copper concentration (R _s=−0.579, p <0.05) and between the activity of ex-Na⁺/Li⁺ and plasma copper concentration (R _s=−0.508, p<0.05). Plasma copper concentration significantly influences the activity of sodium transporting systems in erythrocyte membrane. Copper supplementation could be expected to provide therapeutic benefits for hypertensive patients.     

Spring phytoplankton assemblages in the Southern Ocean between Australia and Antarctica

Variations of phytoplankton assemblages were studied in November–December 2001, in surface waters of the Southern Ocean along a transect between the Sub-Antarctic Zone (SAZ) and the Seasonal Ice Zone (SIZ; 46.9°–64.9°S; 142°–143°E; CLIVAR-SR3 cruise). Two regions had characteristic but different phytoplankton assemblages. Nanoflagellates(<20 μm) and pico-plankton (∼2 μm) occurred in similar concentrations along the transect, but were dominant in the SAZ, Sub-Antarctic Front (SAF), Polar Front Zone (PFZ) and the Inter-Polar Front Zone (IPFZ), (46.9°–56.9°S). Along the entire transect their average cell numbers in the upper 70 m of water column, varied from 3 × 10⁵ to 1.1 × 10⁶ cells l⁻¹. Larger cells (>20 μm), diatoms and dinoflagellates, were more abundant in the Antarctic Zone-South (AZ-S) and the SIZ, (60.9°–64.9°S). In AZ-S and SIZ diatoms ranged between 2.7 × 10⁵ and 1.2 × 10⁶ cells l⁻¹, dinoflagellates from 3.1 × 10⁴ to 1.02 × 10⁵ cells l⁻¹. A diatom bloom was in progress in the AZ-S showing a peak of 1.8 × 10⁶ cells l⁻¹. Diatoms were dominated by Pseudo-nitzschia spp., Fragilariopsis spp., and Chaetoceros spp. Pseudo-nitzschia spp. outnumbered other diatoms in the AZ-S. Fragilaropsis spp. were most numerous in the SIZ. Dinoflagellates contained autotrophs (e.g. Prorocentrum) and heterotrophs (Gyrodinium/Gymnodinium, Protoperidinium). Diatoms and dinoflagellates contributed most to the cellular carbon: 11–25 and 17–124 μg C l⁻¹, respectively. Small cells dominated in the northern region characterized by the lowest N-uptake and new production of the transect. Larger diatom cells were prevalent in the southern area with higher values of N-uptake and new production. Diatom and nanoflagellate cellular carbon contents were highly correlated with one another, with primary production, and productivity related parameters. They contributed up to 75% to the total autotrophic C biomass. Diatom carbon content was significantly correlated to nitrate uptake and particle export, but not to ammonium uptake, while flagellate carbon was well correlated to ammonium uptake, but not to export. Diatoms have contributed highly to particle export along the latitudinal transect, while flagellates played a minor role in the export.     

Effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii (Cyanophyta)

The effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii were determined. Of the three temperatures tested, 10, 25 and 35°C, the maximal geosmin concentration and geosmin productivity were yielded at 10°C, while the highest chl a production was observed at 25°C. In the studies on light intensity, the maximal geosmin concentration and geosmin productivity were observed at 10 μmol m⁻² s⁻¹, while the highest chl a production was at 20 μmol m⁻² s⁻¹. It was suggested that more geosmin was synthesized with lower chl a demand. Meanwhile, the relative amounts of extra- and intracellular geosmin were investigated. Under optimum growth conditions (20 μmol m⁻² s⁻¹, 25°C; BG-11 medium), the amounts of extracellular geosmin increased as the growth progressed and reached the maximum in the stationary phase, while the intracellular geosmin reached its maximum value in the late exponential phase, and then began to decline. However, under the low temperature (10°C) or light (10 μmol m⁻² s⁻¹) conditions, more intracellular geosmin was synthesized and mainly accumulated in the cells. The proportions of extracellular geosmin were high, to 33.33 and 32.27%, respectively, during the stationary phase at 35°C and 20 μmol m⁻² s⁻¹. It was indicated that low temperature or light could stimulate geosmin production and favor the accumulation of geosmin in cells, while more intracellular geosmin may be released into the medium at higher temperatures or optimum light intensity.     

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.     

Beneficial effects of soy protein in the initiation and progression against dimethylbenz [a] anthracene-induced breast tumors in female rats

Objective: Although recent studies link altered cellular redox state to protein dysfunction in various disease-states, such associations are least studied in clinical diabetes. Therefore, this study assessed the levels of reduced glutathione (GSH) and Na⁺/K⁺ ATPase activities in type 2 diabetic patients with and without microangiopathy. Methods: The study group comprised of a total of 160 subjects, which included non-diabetic healthy controls (n = 40) and type 2 diabetic patients without (n = 60) and with microangiopathy (n = 60), defined as presence of retinopathy with or without nephropathy. Erythrocyte Na⁺/K⁺ ATPase activity and GSH levels were estimated spectrophotometrically and fluorometry was used to determine the plasma thiobarbituric acid reactive substances (TBARS) and serum advanced glycation end products (AGEs). Results: GSH levels in diabetic subjects without (4.8± 0.15 μmol/g Hb) and with microangiopathy (5.2± 0.14 μmol/g Hb) were significantly lower (p < 0.001) compared to control subjects (6.3± 0.14 μmol/g Hb). Erythrocyte Na⁺/K⁺ ATPase activity was significantly reduced (p < 0.001) in diabetes subjects with (272± 7 nmol Pi/mg protein/h) and without microangiopathy (304 ± 8) compared to control (374 ± 6) subjects. TBARS were significantly higher (p < 0.001) in diabetes subjects with (10.65± 0.81 nM/ml) and without microangiopathy (9.90± 0.5 nM/ml) compared to control subjects (5.18± 0.18 nM/ml). Advanced glycation end product levels were also significantly (p < 0.001) elevated in diabetic subjects with microangiopathy (8.2± 1.8 AU) when compared to diabetes subjects without microangiopathy (7.0± 2.0 AU) and control subjects (4.6± 1.9 AU). On multivariate regression analysis, GSH levels showed a positive association with the Na⁺/K⁺ ATPase activity and negative association with TBARS and AGE levels. Conclusion: Hypoglutathionemia and increased oxidative stress appears to be early biochemical aberrations in diabetes, and through protein alterations, oxidative stress and redox modifications may contribute to pathogenesis of diabetic microangiopathy.     

Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense rupr.

Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵−6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.     

Arctic Sea ice biota: design and evaluation of a mesocosm experiment

A mesocosm experiment (enclosure volume 220 l) was designed such that sea ice inhabited by Arctic Sea ice organisms was formed and maintained under natural conditions at 66°N in Rovaniemi, Finland. The experiment was run from natural freezing in December 1994 to melting in April 1995. The ice was inhabited by diatoms, chlorophyceae, heterotrophic flagellates, ciliates, nematodes and turbellarians. Biomass in the ice, expressed as Chlorophyll a concentration, was 20–110 μg l⁻¹; total cell densities varied from 5 × 10⁶ to 35 × 10⁶ cells l⁻¹. Amongst phototrophic organisms, a succession from a flagellate-dominated community (Chlamydomonas sp.) to a multi-species diatom-dominated community was observed. Typical Arctic species such as Nitzschia frigida and Melosira arctica were present in the ice. Bacterial concentration varied between 2 × 10⁸ and 7 × 10⁸ cells l⁻¹. At least two trophic levels were present in the ice. Received: 3 April 1997 / Accepted: 9 September 1997     

Effect of Resuscitation with 21% Oxygen and 100% Oxygen on NMDA Receptor Binding Characteristics Following Asphyxia in Newborn Piglets

The present study investigated the effect of reventilation with 21% and 100% oxygen following asphyxia in newborn piglets on NMDA receptor binding characteristics, Na⁺, K⁺-ATPase activity, and lipid peroxidation. After achieving a heart rate less than 60 beats per minute, asphyxiated piglets were reventilated with 21% oxygen or 100% oxygen. ³[H]MK-801 binding showed the Bmax in the 21% and 100% groups to be 1.53 ± 0.43 and 1.42 ± 0.35 pmol/mg protein (p = ns). Values for Kd were 4.56 ± 1.29 and 4.17 ± 1.05 nM (p = ns). Na⁺, K⁺-ATPase activity in the 21% and 100% groups were 23.5 ± 0.9 and 24.4 ± 3.9 μmol Pi/mg protein/h (p = ns). Conjugated dienes (0.05 ± 0.02 vs. 0.07 ± 0.03 μmol/g brain) and fluorescent compounds (0.54 ± 0.05 vs. 0.78 ± 0.19 μg quinine sulfate/g brain), were similar in both groups (p = ns). Though lipid peroxidation products trended higher in the 100% group, these data show that NMDA receptor binding and Na⁺, K⁺-ATPase activity were similar following reventilation with 21% or 100% oxygen after a single episode of mild asphyxia. Funded in part by a grant from the American Academy of Pediatrics/American Heart Association/Neonatal Resuscitation Program and NIH grant number HD-20337.     

CCN3 suppresses mitogenic signalling and reinstates growth control mechanisms in Chronic Myeloid Leukaemia

CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia (CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 10⁶ copies per 50 ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within the subG₀ phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p < 0.05). K562/CCN3 cells exposed to Imatinib (1 μM and 5 μM) showed an increase in events within the subG₀ phase of cell cycle over 96 h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells treated with recombinant human Ccn3 (10 nM) in combination with Imatinib (5 μM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control) which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase in gene expression respectively (n = 3, p < 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies.     

Regulation of the Expression of the Photosynthetic Apparatus of Rhodobacter capsulatus Grown in Nitrogen-Limited Chemostat Cultures

Rhodobacter capsulatus was grown in chemostat cultures under different dilution rates and with ammonium ions as the limiting nutrient. The maximal growth rate (μmax) and the Monod cell growth saturation coefficient (K_s), were calculated from batch cultures grown at different concentrations of NH₄ ⁺. The experiments in chemostat were carried out at 0.25 mM (NH₄)₂SO₄, and the dilution rates were varied between 38% and 75% of μmax. The results indicated that under continuous culture conditions the cell yield coefficient (Y) (mg dry weight × μmol consumed ammonium sulfate⁻¹) decreased with increasing dilution rate (D). On the contrary, the cell yield was constant when expressed as mg cellular protein ×μmol consumed ammonium sulfate⁻¹. This occurred as a consequence of both an increase in the consumed ammonium sulfate and a simultaneous decrease in the cell biomass production at increasing growth rates. The cells produced at higher growth rates had a higher protein content per cell. The specific content of bacteriochlorophyll (Bchl) decreased (between 3 and 4 times) with increasing growth rates measured in either cells or chromatophores. However, the absorption spectra of the cells indicated that the ratio LHI (light-harvesting complex I) to LHII (light-harvesting complex II) Bchl complexes did not change. The reaction center (RC) complex content varied in parallel with the total Bchl content, yielding a constant photosynthetic unit of 65 mol Bchl × mol RC⁻¹ at different Ds. On the other hand, the uncoupled ATPase-specific activity measured in chromatophores was usually between 30% and 40% higher at the highest growth rates reached in these experiments. Received: 22 January 1996 / Accepted: 9 March 1996     

Optimization of protoplast yields from the red algae <Emphasis Type="Italic">Gracilaria dura</Emphasis> (C. Agardh) J. Agardh and G. <Emphasis Type="Italic">verrucosa</Emphasis> (Huds.) Papenfuss

This study reports on the optimization of protoplast yield from two important tropical agarophytes Gracilaria dura and Gracilaria verrucosa using different cell-wall-degrading enzymes obtained from commercial sources. The conditions for achieving the highest protoplast yield was investigated by optimizing key parameters such as enzyme combinations and their concentrations, duration of enzyme treatment, enzyme pH, mannitol concentration, and temperature. The significance of each key parameter was also further validated using the statistical central composite design. The enzyme composition with 4% cellulase Onozuka R-10, 2% macerozyme R-10, 0.5% pectolyase, and 100 U agarase, 0.4 M mannitol in seawater (30‰) adjusted to pH 7.5 produced the highest protoplast yields of 3.7 ± 0.7 × 10⁶ cells g⁻¹ fresh wt for G. dura and 1.2 ± 0.78 × 10⁶ cells g⁻¹ fresh wt for G. verrucosa when incubated at 25°C for 4–6 h duration. The young growing tips maximally released the protoplasts having a size of 7–15 μm in G. dura and 15–25 μm in G. verrucosa, mostly from epidermal and upper cortical regions. A few large-size protoplasts of 25–35 μm, presumably from cortical region, were also observed in G. verrucosa.