Cultivation of explants of the marine sponge Crambe crambe in closed systems

Explants of the sponge Crambe crambe were cultured in natural seawater, with or without marine microalga (Phaeodactylum tricornutum) in discontinuous flow through systems and in continuous flow-through systems (DFTHS and CFTHS, respectively). Growth was measured as the increase in underwater weight. In the experiment carried out in the CFTHS, the explants average underwater weight increased by up to 1380% of the initial weight in 22-45 days. Growth in DFTHS was much slower producing a gain of up to an average value of 322% of the initial weight in 100-210 days. Growth kinetics varied considerably for different explants. Explants grew fastest in the first 10-days of subculture. The sponges grew better in CFTHS compared with the DFTHS. The high growth rates observed in CFTHS suggest that this technique is a promising method for culturing C. crambe in closed systems.     

Growth of the sponge Pseudosuberites (aff.) andrewsi in a closed system

Explants of the Indo-Pacific sponge Pseudosuberites aff. andrewsi were fed with the microalgae Chlorella sorokiniana and Rhodomonas sp. It was microscopically observed that these algae were ingested and digested by the sponge cells, suggesting that they were consumed by the sponges. The algae were further used for two growth experiments with five explants of P. aff. andrewsi and four explants of P. andrewsi. Growth was measured as the increase in projected body area. The explants showed considerable growth (up to 730% in 54 days for P. aff. andrewsi and up to 680% in 22 days for P. andrewsi), which is much higher than previously reported growth rates for sponges. Growth started after a stationary phase of 5–20 days in which the projected body area did not increase. The growth of P. aff. andrewsi appeared to be linear and was inhibited at the end of the experiment. Two explants of P. andrewsi showed exponential growth instead of linear growth. Hence, no general statements about the growth kinetics of these sponges can be made at this time. However, the high growth rates found in this study suggest a promising future for cultivation of sponges in closed systems.     

In Vitro CuIture of the Tropical Sponge <Emphasis Type="Italic">Axinella corrugata</Emphasis> (Demospongiae): Effect of Food Cell Concentration on Growth,Clearance Rate,and Biosynthesis of Stevensine

In vitro culture is one possible method for supplying sponge metabolites for pharmaceutical applications, but appropriate feeding regimens that maximize both growth and metabolite biosynthesis are largely unknown. According to the natural concentration (NC) of cells 1 to 50 µm in size that are available to wild Axinella corrugata, we fed explants a multispecific diet of bacteria, microalgae, and yeast at 4 different concentrations: 1NC, 3NC, 5NC, and 5+1NC (the last consisted of 5 NC of bacteria and 1 NC of microalgae and yeast). Explants fed a 3NC diet had the best culture response, growing on average from 8.5 g to 10.3 g in 8 weeks, and showing a 110% increase in concentration (milligrams per gram of dry weight) of the antitumor compound stevensine. Stevensine production in 3NC explants, representing the total milligrams of metabolite per explant, increased by 157% over the study. Explants fed at 1NC had relatively stable weights, indicating that the diet met metabolic costs only. Explants fed at the two highest concentrations lost weight after 4 weeks, possibly because long-term high cell concentration blocked their aquiferous system, reducing their ability to feed efficiently. Stevensine production in explants fed the 1NC, 5NC, or 5+1NC diets were similar, and varied little from the initial amount. A separate experiment showed that the clearance rate for A. corrugata is similar between the examined food types and cell concentrations over 5 hours, averaging 766 ml h^–1 g DW^–1.Overall, this study demonstrates that relatively small changes in food abundance can greatly affect both sponge growth and metabolite biosynthesis. The good growth and increased production of the target metabolite stevensine for A. corrugata explants fed a 3NC diet suggests that in vitro culture is a viable method of supplying some sponge metabolites.     

Long-term culture of sponge explants: conditions enhancing survival and growth,and assessment of bioactivity

Sponges are an important source of secondary metabolites with pharmaceutical interest. This is the main reason for the increasing interest of sponge culture recent years. The optimal culture system depends on the species to be cultured: while some species easily produce sponge aggregates after dissociation (primmorphs), others show a great capacity to regenerate after fragmentation (explants). Corticium candelabrum is a Mediterranean bacteriosponge that can undergo asexual reproduction. We have taken advantage of this capability and cultured C. candelabrum explants under several experimental conditions. To find the best conditions for obtaining functional explants, we assayed a range of conditions, including seasons of collection, culture temperature, filtered versus filtered-sterile seawater, addition of antibiotics and proportion of ectosome. We monitored the changes in shape and ultrastructure during the formation of explants. After 24 h, TEM images showed the aquiferous system disarranged, in particular at the sponge periphery. From 2 to 4 weeks later, the aquiferous system regenerated, and fragments became functional sponges (explants). Explants were cultured under two regimes: in vitro and in a closed aquarium system. Antibiotics were only added to the in vitro culture to assess their effect on the symbiotic bacteria, which remained healthy despite the presence of antibiotics. Two food regimens (marine bacteria and green algae) were assayed for their ability to satisfy the metabolic requirements of explants. We monitored explant survival and growth. Explants showed a high long-term survival rate (close to 100%). Growth rates were higher in the closed aquarium system, without antibiotic addition, and fed with algae. Explants cultures were hardly contaminated because manipulation was reduced to a minimum and we used sterilized seawater. C. candelabrum produces bioactive molecules, which may play a defensive role in the sponge and may have pharmaceutical interest. The bioactivity of the explants was similar to that of wild sponges.     

Antifeedant and toxic activity of Adhatoda vasica leaf extract against Spodoptera littoralis (Lep., Noctuidae)

Abstract: The effect of crude methanolic extracts of Adhatoda vasica leaves on the feeding and performance of Spodoptera littoralis larvae was investigated in the laboratory. Feeding on fresh leaves resulted in 100% mortality of larvae after 26 days of unsubstantial growth. The extract exhibited strong antifeedant and toxic activity against the larvae when applied either on leaf discs or incorporated into artificial diet. Under choice conditions the antifeedant index calculated over 72 h for neonate larvae increased significantly (from 71.5 ± 3.2 to 92.1 ± 4.2) as the concentration of extract in the treated diet increased from 200 to 1000 ppm. Consumption by the sixth instar larvae of leaf discs dipped in 0.01, 0.1 and 0.2% extract solutions was significantly lower than consumption of control discs in both choice and no-choice tests. The latter two concentrations deterred feeding by 63.4 and 90.4%, respectively, under choice conditions, while only the 0.2% extract solution deterred feeding by 56.8% in the no-choice test. Toxicity of the extracts was manifested by a high mortality, reduced growth rates, and low weight gain by larvae fed on diets containing 200–2000 ppm of the extract. No larvae survived to pupation under the latter concentration. The time to pupation increased from 15.8 ± 0.4 to 37.9 ± 4.1 days as the extract concentration in diet increased from 0 to 1000 ppm. When fed to the fifth instar larvae, the crude extract significantly reduced consumption, growth, utilization of ingested and digested food, and approximate digestibility. The consumption-dependent growth efficiency of animals fed on extract-free diet was significantly higher than the growth efficiency of animals fed on extract-containing diets, suggesting both antifeedant and toxic activities of the extract.     

Feeding history and obese-prone genotype increase survival of rats exposed to a challenge of food restriction and wheel running

We hypothesized that obese-prone genotype and history of food restriction confer a survival advantage to genetically obese animals under environmental challenge. Male juvenile JCR:LA-cp rats, obese-prone and lean-prone, were exposed to 1.5 h daily meals and 22.5-h voluntary wheel running, a procedure inducing activity anorexia (AA). One week before the AA challenge, obese-prone rats were freely fed (obese-FF), or pair fed (obese-PF) to lean-prone, free-feeding rats (lean-FF). Animals were removed from protocol at 75% of initial body weight (starvation criterion) or after 14 days (survival criterion). AA challenge induced weight loss in all rats, but percent weight loss was more rapid and sustained in lean-FF rats than in obese-FF or obese-PF animals (P < 0.04). Weight loss was significantly higher in obese-FF rats than obese-PF rats, 62% of which achieved survival criterion and stabilized with zero weight loss. Obese-PF rats survived longer, on average (12.0 ± 1.1 day) than obese-FF (8.2 ± 1.1 day) and lean-FF rats (3.5 ± 0.2 day) (P < 0.02). Wheel running increased linearly in all groups; lean-FF increased more rapidly than obese-FF (P < 0.05); obese-PF increased at an intermediate rate (P < 0.02), and those rats that survived stabilized daily rates of wheel running. Prior food restriction of juvenile obese-prone rats induces a survival benefit beyond genotype, that is related to achievement of homeostasis. This metabolic adaptive process may help explain the development of human obesity in the presence of an unstable food environment which subsequently transitions to an abundant food supply.     

ATP N-glycosidase - a novel ATP-converting activity from a marine sponge Axinella polypoides.

A novel nucleosidase enzymatic activity was discovered in the marine sponge Axinella polypoides. This enzyme, designated as ATP N-glycosidase, converts adenosine-5'-triphosphate into adenine and ribose-5-triphosphate. The crude extract of A. polypoides was capable of hydrolysing 25 micro mol ATP.min-1 per g wet weight of sponge. The catalytic activity of a sponge crude extract per mg total protein is comparable with specific activities of purified plant adenosine and bacterial AMP nucleosidases. The preferred substrate for the novel enzyme is ATP but any compound containing adenosine-5'-diphosphoryl fragment is also cleaved. The biochemical properties (Km, Kip, environmental requirements) of ATP N-glycosidase show similarities with previously described adenine-specific nucleosidases; however, the pattern of its biochemical characteristics does not match with that of any of those enzymes.     

The effect of chronic food and water restriction on open-field behaviour and serum corticosterone levels in rats

In operant conditioning experiments, two methods are commonly used to motivate laboratory rats to perform designated tasks. The first is restricting food so that rats are forced to lose 20% of body weight within one week, followed by maintenance at 80% of the baseline weight for the remainder of the experiment. The second is restricting access to water to 15 min in each 24 h period. These methods are effective in motivating the animals. There is, however, little information available on the effects on performance in tests of behaviour that are not related to operant conditioning. In addition, it is not clear if these commonly used methods of food and water restriction will lead to physiological stress as indicated by an elevation of serum corticosterone. Male rats were either food-restricted to reduce and maintain their weight at 80% of baseline weight, or were restricted to 15 min access to water every 24 h. Activity in the open field was significantly greater in food-restricted rats than in water-restricted or control rats, but freezing behaviour was similar in all experimental groups. Food-restricted rats had a higher mean serum corticosterone level than water-restricted and control rats 37 days after the start of the experimental period. These data suggested that chronically restricting food and maintenance of body weight at 80% of baseline body weight led to significant behavioural changes and physiological stress. In contrast, water restriction did not lead to changes in behaviour or corticosterone levels. A second experiment was conducted to compare the effects of food restriction to 80% of baseline body weight, as described above, with a less stringent protocol in which test rats were initially reduced to 80% of baseline weight, but were then maintained at 80% of an ad libitum fed control rat's weight. Serum corticosterone levels and adrenal gland weights were measured after the initial week of forced weight loss and after maintenance for 21 days. Forced loss of 20% of body weight in the first week led to significantly increased serum corticosterone levels and adrenal gland weights compared to ad libitum fed controls. Serum corticosterone levels and adrenal gland weights in rats maintained at 80% of their initial body weight for 21 days remained higher than ad libitum fed control rats. However, rats maintained at 80% of an ad libitum fed control rat's weight did not differ from control rats in serum corticosterone levels or adrenal gland weights at the end of the 21-day study period. Adjustment of the feeding regimen in this manner eliminated physiological evidence of chronic stress.     

Effect of food type and concentration on the survival,longevity, and reproduction of Daphnia magna

The freshwater microalgae Ankistrodesmus falcatus and Scenedesmus incrassatulus, at three concentrations (6, 12 and 18 mg l⁻¹ t, dry weight), were tested as diets for Daphnia magna cultured in reconstituted hard water, at 19 ± 1 °C. Effects on survival, and reproductive characteristics were evaluated during a complete life-cycle. Test animals fed with the highest food concentration had the lowest survival, longevity, number of clutches, and average inter-brood times, but the total offspring was similar to that obtained with the lowest food concentration, for the same species of alga. Average longevity ranged from 40 to 85 days, with a minimum of 24 and a maximum of 119 days. The average inter-brood time ranged from 4.2 to 5.8 days, the highest values being observed at the lowest food concentration. The average total offspring was maximum for the intermediate concentrations of both algae. The maximum number of clutches ranged from 9 to 23, with the lowest values being observed at the highest food concentration. Even though both microalgae had similar effects on survival and reproduction, and the greatest differences observed were related to food concentration, it seems that S. incrassalulus is a comparatively better food for D. magna. The most suitable food level should be determined prior to carrying out chronic bioassays, and it is also an important factor in cultures for obtaining neonates for toxicological tests.     

The role of hormones and gradients in the initiation of cortex proliferation and nodule formation in Pisum sativum L.

Summary The effect of exogenous phytohormones on proliferation of the root cortex, and their relation to the division factors from Rhizobium which participate in the initiation of root nodules, were studied using explants of root-cortex tissue from 7-day-old, sterile pea plants. The explants were cultured for 7 days on a synthetic nutrient medium supplemented with auxin, or auxin and cytokinin. With only auxin present in the medium, ca. 10% of the explants showed cell proliferation. With both auxin and cytokinin this percentage was much higher (ca. 80%). The active explants showed proliferation patterns which were similar to or could be derived from a pattern with three predominant meristematic areas in the inner cortex opposite the three xylem radii of the excised central cylinder. These proliferation patterns were similar to the initial proliferative stages in root-nodule formation in seedling intact roots. From this restricted division response of the explants to the hormones, a hypothesis of endogenous division factors is proposed. To test this hypothesis, extractions of root tissue were performed. The addition of a crude alcoholic extract from the central cylinder or the cortex to the medium resulted in cell divisions throughout the cortex. The results are interpreted as evidence for the presence of a transverse gradient system of (an) unknown cell-division factor(s) in the root cortex which may control the induction of cell divisions in nodule initiation brought about by the release of auxin and cytokinin from Rhizobium.     

Resource allocation in Tomocerus flavescens (Insecta,Collembola): a study with C-14-labelled food

Summary Individuals of the collembolan species Tomocerus flavescens from a beech wood on limestone near Göttingen (West Germany) were fed with C-14-labelled algae in the laboratory. On an average, T. flavescens exchanged almost all of its endogenous carbon after 3.4 moults. In periods of starvation the interval between two ecdyses increased from 5.2 to 6.1 days (17°C). The assimilation efficiency was determined in three independent ways (carbon use of different algal components; A=C-FU; C=P+R+FU, A=P+R): it ranged from 0.34 to 0.40 (converted to energy values 0.30 to 0.45). It could be raised in phases of high mobility and in periods of food shortage by lowering the gut passage rate. The decrease in feeding activity (demonstrated by gut contents analyses) in case of a detoriation of the food quality and the food availability can be partly compensated in this way. For individuals which did not reproduce, the ratio of production to assimilation was 0.24 (converted to energy values 0.31). T. flavescens showed an increased body growth in connection with reaching sexual maturity. Specific demands of nutritive substances in periods of physiological changes could possibly be compensated by a more efficient resource use as well as by differences in resource allocation. The studied population of T. flavescens was compared both with that of a Danish beech forest and general data from the literature. The production of few eggs rich in energy, the high weight of newly hatched individuals, the high growth as well as the storage of carbon connected with egg production, the comparatively low respiration metabolism and the high amount of energy invested in search for food have to be regarded as an adaptation to the habitat. According to conservative estimates climbing individuals of this species consume 0.83 g of the algal dry mass growing on one beech tree during one summer. T. flavescens mainly uses the ethanol-soluble components of the algae.     

Green urchin as a significant source of fecal particulate organic matter within nearshore benthic ecosystems

The role of green sea urchin Strongylocentrotus droebachiensis as a source of fecal particulate organic matter (POM) for the benthic nearshore ecosystems has been studied over a 3.5-month period. Three macroalgae were tested as food sources: Alaria esculenta, Laminaria longicruris and Ulvaria obscura. Urchins were fed ad libitum with either a single alga species or a mixture of all three algae. Consumption and defecation rates were determined as well as the feces/alga ratio in term of biomass and biochemical composition. Consumption rate increased exponentially with urchin size and also varied with alga species. In the single alga trial, consumption rate was higher for both brown algae (Laminaria and Alaria) compared to Ulvaria. Urchins feeding on the mixture of algae maintained their total ingestion rate (sum of the three algae) at the same level to those feeding on a single alga diet. The mixed algae trial showed that urchins clearly preferred Laminaria (72% of total ingestion) over Alaria (22%) and Ulvaria (6%). The defecation rate was tightly correlated with the food consumption rate and thus increased with urchin size. On average, 75% of the ingested algal biomass was released as fecal POM. The percentage of food defecated changed with alga species, with the highest value for Alaria (81%) and the lowest for Laminaria (67%). The percentage of food defecated by urchins feeding on the mixture of algae was generally comparable to those feeding on single alga diet. Biochemical composition (in soluble carbohydrates, proteins and lipids) of urchin fecal POM reflected that of the algae content. From 40% to 80% of macronutrients in algal food persisted in fecal matter. This proportion varied with the alga species and macronutrient considered. This study shows that the green sea urchin plays a significant role in the production of POM within nearshore benthic ecosystems, and it is a potentially nutritious food source for detritivores.     

Effects of food deprivation on the development of Coleomegilla maculata (De Geer) (Coleoptera: Coccinellidae)

The lady beetle Coleomegilla maculata (De Geer) is a natural enemy of several insect pests and feeds on pollen and nectar to survive periods when prey is scarce. The effect of the feeding interval on the development, survival, fecundity, and longevity of C. maculata was determined. Newly hatched larvae of C. maculata were reared individually and fed with eggs of the Mediterranean flour moth Anagasta kuehniella (Zeller) at intervals of one, two, and three days under controlled conditions (23 ± 1oC; 60 ± 10% RH; 12 h phtophase). The duration of larval instars and the total larval stage was prolonged as the feeding interval increased. The larval period lasted on average 9.2 ± 0.19 days when the larvae were fed daily with prey, and 14.6 ± 0.48 days when food was offered at three-day intervals. There was an inverse relationship between food intervals, survival, and weight of larvae and adults of the coccinellid. Survival rate of larvae fed daily was 76.8%, while the rate was 50.0% and 23.4% for larvae fed every two and three days, respectively. Coleomegilla maculata showed fecundity of 781.1 ± 149.02, 563.4 ± 80.81 and 109.0 ± 103.0 eggs when fed daily and at intervals of two and three days, respectively.     

Actinidia deliciosa C.F. Liang in vitro

In vitro growth of Actinidia deliciosa C.F. Liang, cv Hayward and changes in mineral composition of the medium and in the different parts of the explants (callus, stem and leaves), were analyzed after 0, 15, 30, 45 and 60 days of culturing in each of three successive 60 days subcultures.Fresh (FW) and dry weight (DW) of the explants increased mainly during the first 30 days of culturing, with a predominant increase of FW in leaves and an equal distribution in DW in callus and leaves. Stem FW and DW changes were lower than those observed with callus and leaves. As FW and DW of the explants increased the FW and DW of the medium decreased.The presence of the explants induced a large decrease of medium pH during the first 15 days of culturing followed by a return to the initial level at the end of the culturing.The initial P content of the MS medium was insufficient for the long term culturing, as after 30 days of culturing almost all (94.5%) the P present in the medium was absorbed by the explants and evenly distributed in their different parts. During the first month, 85% of the initial N was absorbed. At the end of the culture only 2% of the initial P and 5% of N remained in the medium. These two elements were equally distributed in callus and leaves during the first month of culturing, while in the last 30 days they increased only in the callus.MS medium initial concentrations of K, Mg, Ca, Fe, Zn, Mn and Cu were sufficient for 60 days explants growth. Almost all these elements were absorbed during the first 30 days of culturing. Their distribution in the different parts of the explant was uneven throughout the culture period. Callus tissue was the main site for accumulation of all these mineral elements.     

Effects of caecectomy in the young adult female rat on digestibility of food offered ad libitum and in restricted amounts.

Caecectomized and sham-operated rats were fed a laboratory chow ad libitum and the effects of caecectomy on the digestibility of the food were studied. Compared with sham-operated controls, caecectomized rats showed a decrease in apparent digestibiltiy of organic matter from 77.8 to 73.0%, of crude protein from 83.0 to 79.4%, and of 'carbohydrate' from 74.6 to 69.0%. However, faecal water content increased from 41.6 to 54.8%. 51Cr-labelled EDTA was excreted faster in the faeces after caecectomy. The colon partly adapted to the loss of caecal mucosa by increased length and thus mucosal surface area. In a second concurrent experiment the effect of caecectomy on the apparent digestibility of food during food restriction was studied. Six caecectomized rats, comparable in all respects to those used in the first study, were fed the laboratory chow ad libitum for 3 weeks. They were then fed submaintenance amounts of food to achieve body weight losses of 40--50% and to maintain these low weights for 4 weeks. Finally, they were again fed ad libitum for 3 weeks. During the period of restriction the apparent digestibility of organic matter increased from 72.7 to 75.4%. This was largely due to the increased apparent digestibility of crude protein which rose from 78.4 to 81.9%. Digestibility coefficients returned to control values immediately upon refeeding ad libitum.     

Dietary GABA decreases body weight of genetically obese mice

Body weight and food intake were decreased more in lean than in genetically obese (ob/ob) mice fed a low protein diet containing GABA. Young lean and obese mice, when fed a 4.5% GABA diet, lost 30% or 20% of their initial weight over periods of 10 or 32 days, respectively. When refed the control diet both groups of mice gained weight rapidly but the obese grew more over a longer period of time. With GABA at 2 or 2.5% of the diet, respectively, lean and obese mice could be maintained at constant low weights for weeks. The obese were less sensitive than the lean mice to dietary GABA. Weights increased rapidly when a high protein diet containing GABA was fed. Hepatic GABA aminotransferase activity increased in all mice fed the high protein diet.     

Effects of feeding on survival, cannibalism, growth and feed conversion of African catfish, Clarias gariepinus (Burchell) in concrete tanks

Daily feeding rates of 0, 2, 4, 6, 8 and 10% of body weight to juvenile (average weight 0.64–65.4 g) catfish, Clarius gariepinus, for 112 days had significant effects on survival, cannibalism, growth and feed conversion ratio (FCR). Survival was significantly higher in fish fed at 6–10% of body weight than those fed at 2–4%. Mortality was mainly caused by cannibalism, being significantly higher in underfed (2–4%) fish than in fish fed at the 6–10% level. A feeding level above 6% did not further reduce cannibalism. The best growth performance was recorded for the fish fed at 8–10% body weight, followed by the fish fed at 4–6%; poorest growth was found for those fed at the 2% level. FCR significantly increased from 2–4% (FCR, 1.14–1.13) to 6, 8 and 10% (FCR, 1.37, 2.18, 2.98, respectively). The results of this study suggest that the optimum feeding rate of juvenile catfish with an average initial weight of about 0.64 g and grown to a size of about 64 g is at 6% body weight per day.     

Nutrient translocation from green algal symbionts to the freshwater sponge Ephydatia fluviatilis

The symbiosis between the freshwater sponge Ephydatia fluviatilis and a chlorella-like green alga is not obligate and only occurs when the sponge grows in the light. The algae accumulate intracellular pools of sucrose and glucose and translocate between 9 and 17% of the total photosynthate to the host. The principal product translocated is glucose which is fed directly into the sponge metabolic pool. White sponges transplanted back into the river in the shade grew logarithmically with a mean doubling time of 12 days. Sponges transplanted into illuminated habitats did not grow. It is unknown how the sponge acquires its algal symbiont.     

Influences of crude extract of tea leaves,Camellia sinensis,on streptozotocin diabetic male albino mice

Natural remedies from medicinal plants are considered to be effective and safe alternative treatment for diabetes mellitus. The aim of the present study was to investigate the hypoglycemic activity of the crude tea leaves extract on streptozotocin (STZ)-induced diabetic mice. The average body weight of animals with diabetes and their percentage changes of body weight gain after 15 and 30 days were significantly lower than that of the normal control mice. In diabetic mice, supplementation with tea leaves extract decreased the loss of body weight. After 15 and 30 days, significant increases in the levels of serum glucose, triglycerides, cholesterol, creatinine, urea, uric acid, glutamic pyruvic acid transaminase (GPT) and glutamic oxaloacetic acid transaminase (GOT) were noted in STZ-diabetic mice fed with normal diet. Also, the values of total protein in this group were statistically declined after 15 and 30 days. The levels of serum glucose and GPT were significantly elevated after 15 and 30 days in diabetic mice supplemented with tea leaves extract. Moreover, the level of serum GOT was notably increased after 30 days. Insignificant alterations were observed in the levels of serum triglycerides, cholesterol, total protein, creatinine, urea and uric acid in diabetic mice supplemented with tea leaves extract. Thus, the present results have shown that tea leaves extract has the antihyperglycemic, antihyperlipidemic, and antihyperproteinemic effects and consequently may alleviate liver and kidney damage associated with STZ-induced diabetes in mice.     

Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L-15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L(-1)) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v) resulted in an evident sustained long-term growth of C. crambe explants. This growth enhancement produced high metabolic activity in the explants, as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose. The lactate accumulation increased with the size and age of explants. Prior to these experiments, we successfully developed a robust new alternative method, based on digital image treatment, for accurate determination of the explant apparent volume as growth measure.