Trichloroacetic Acid-insoluble Fraction Research Articles

Characterization of physicochemical parameters and the effect on the labeling of blood constituents with technetium-99m of a Solanum melongena commercial extract

This investigation evaluated physicochemical parameters and the effect on the labeling of blood constituents with technetium-99m of a Solanum melongena aqueous extract. A commercial S. melongena was used to prepare an aqueous extract and visible absorbance spectrum, electric conductivity and refractive index were determined. Blood samples fromWistar rats were incubated with S. melongena extract or with 0.9% NaCl solution as control. After, stannous chloride as reducing agent and technetium-99m as sodium pertechnetate were added. Plasma and blood cells samples were separated. Samples of plasma and blood cells were also precipitated with trichloroacetic acid and soluble and insoluble fractions were separated. The radioactivity in each fraction was counted and the percentage of radioactivity incorporated was calculated. Data showed an absorbance peak at 500 nm and electric conductivity and refractive index were higher at the highest extract concentrations. S. melongena extract did not interfere on the labeling of blood constituents with technetium-99 m. The determination of these physicochemical parameters would contribute to characterize a S. melongena aqueous extract. Moreover, as the labeling of blood constituents with technetium-99m depends on the presence of reducing agent, the absence of effect of this extract in this radiolabeling process could be associated with its redox properties. Key words: Solanum melongena, eggplant, labeling, technetium-99 m, physicochemical parameters.

Concentration and time-dependent inter-relationships for cytotoxicities of nitrogen mustard drugs against lymphoblasts in-vitro.

Peripheral lymphoblasts were exposed either to different initial concentrations of the alkylating agents (melphalan, chlorambucil or phenylacetic acid mustard) using a fixed incubation time or a constant [3H]methylthymidine incorporation into the trichloracetic acid-insoluble fraction of the cells. The concentration-time relationships were evaluated by calculating the amount of drug which had chemically reacted in the incubation system. Melphalan showed lower cytotoxicity at short exposure times and high drug concentrations, while chlorambucil exhibited higher cytotoxicity at longer exposure times. In the latter case the effect could be accounted for by the cytotoxic activity of monohydroxy chlorambucil which was formed in the incubation system.

Evaluation of thein vitroeffect of aLantana camaraextract on the labeling of blood constituents of rats with technetium-99m

Blood constituents labeled with technetium-99m (99mTc) have been used in nuclear medicine procedures and drugs are capable to interfere on this labeling. Lantana camara (lantana) has medicinal properties and it has been used in folk medicine. The aim is to verify the effect of a lantana extract on the labeling of blood constituents with 99mTc. Blood of rats was incubated with extract, stannous chloride and 99mTc, as sodium pertechnetate. Plasma (P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) were separated. The % of radioactivity (%ATI) in these samples was calculated. Samples of labeled BC were washed and the %ATI maintained (%ATI-M) in the BC was determined. The results showed that lantana extract decreased significantly (p < 0.05) in the IF-P from 70.24 +/- 2.59 to 11.95 +/- 3.07. This effect was not observed in the BC and IF-BC. The BC-%ATI-M was significantly (p < 0.05) decreased in all concentrations tested when the BC was washed. This fact was not observed in the control. Substances present on the extract should have redoxi action decreasing the concentration of the stannous ion and this condition could justify the effect on the IF-P. The results about the BC-%ATI-M should indicate a possible effect on the transport of ions through the erythrocyte membrane.

The Analysis of the Effect of a Chayotte Extract on the Radiolabeling of Blood Elements in Diabetic Rats

The use of natural products occurs around the world. The labeling of blood constituents with technetium-99m (99m Tc) has been influenced by natural extracts and oxidative stress. Some studies suggested that maternal diabetes can affect the embryology environmental and this fact could help to elucidate that the oxidative stress may be related to the disturb of the gene expression which is essential in the control of the ontogenetic processes. We evaluated the influence of a chayotte(Sechium edule) extract on the labeling of blood elements with 99m Tc in diabetic female rats. The animals were treated with chayotte during 7 days and samples of blood were withdrawn. The samples were incubated with stannous chloride and with 99mTc. Plasma(P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) separated. The radioactivity (ATI%) was rated in RBC, IF-P and IF-C in a well counter. In the diabetic group it was observed an increase in the radioactivity in BC (from 43.65 ± 1.83 to 59.47 ± 1.83 and in the IF-BC (from 26.22 ± 23.58 to 73.28 ± 23.58). It was noticed that the referred extract has normalizing the efficiency of radiolabeling in the diabetic animals which have received the referred extract. The effect of chayotte extract probably, could be explained by the metabolization of the chayotte that would be capable of inducing the generation of active metabolites with oxidant properties probably altering the activity of cell membrane.

Regulation of Intracellular Accumulation of Mutant Huntingtin by Beclin 1

Intracellular accumulation of mutant Huntingtin with expanded polyglutamine provides a context-dependent cytotoxicity critical for the pathogenesis of Huntington disease (Everett, C. M., and Wood, N. W. (2004) Brain 127, 2385-2405). Here we demonstrate that the accumulation of mutant Huntingtin is highly sensitive to the expression of beclin 1, a gene essential for autophagy. Moreover, we show that the accumulated mutant Huntingtin recruits Beclin 1 and impairs the Beclin 1-mediated long lived protein turnover. Thus, sequestration of Beclin 1 in the vulnerable neuronal population of Huntington disease patients might further reduce Beclin 1 function and autophagic degradation of mutant Huntingtin. Finally, we demonstrate that the expression of beclin 1 decreases in an age-dependent fashion in human brains. Because beclin 1 gene is haploid insufficient in regulating autophagosome function (Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y., Cattoretti, G., and Levine, B. (2003) J. Clin. Invest. 112, 1809-1820; Yue, Z., Jin, S., Yang, C., Levine, A. J., and Heintz, N. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15077-15082), we propose that the age-dependent decrease of beclin 1 expression may lead to a reduction of autophagic activity during aging, which in turn promotes the accumulation of mutant Htt and the progression of the disease.

β-Synuclein Reduces Proteasomal Inhibition by α-Synuclein but Not γ-Synuclein

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathogenesis of Parkinson's disease. Recent studies indicate that aggregated alpha-synuclein binds to S6', a component of the 19 S subunit in the 26 S proteasome and inhibits 26 S proteasomal degradation, both ubiquitin-independent and ubiquitin-dependent. The IC(50) of aggregated alpha-synuclein for inhibition of the 26 S ubiquitin-independent proteasomal activity is approximately 1 nm. alpha-Synuclein has two close homologues, termed beta-synuclein and gamma-synuclein. In the present study we compared the effects of the three synuclein homologues on proteasomal activity. The proteasome exists as a 26 S and a 20 S species, with the 26 S proteasome containing the 20 S core and 19 S cap. Monomeric alpha- and beta-synucleins inhibited the 20 S and 26 S proteasomal activities only weakly, but monomeric gamma-synuclein strongly inhibited ubiquitin-independent proteolysis. The IC(50) of monomeric gamma-synuclein for the 20 S proteolysis was 400 nm. In monomeric form, none of the three synuclein proteins inhibited 26 S ubiquitin-dependent proteasomal activity. Although beta-synuclein had no direct effect on proteasomal activity, co-incubating monomeric beta-synuclein with aggregated alpha-synuclein antagonized the inhibition of the 26 S ubiquitin-independent proteasome by aggregated alpha-synuclein when added before the aggregated alpha-synuclein. Co-incubating beta-synuclein with gamma-synuclein had no effect on the inhibition of the 20 S proteasome by monomeric gamma-synuclein. Immunoprecipitation and pull-down experiments suggested that antagonism by beta-synuclein resulted from binding to alpha-synuclein rather than binding to S6'. Pull-down experiments demonstrated that recombinant monomeric beta-synuclein does not interact with the proteasomal subunit S6', unlike alpha-synuclein, but beta-synuclein does bind alpha-synuclein and competes with S6' for binding to alpha-synuclein. Based on these data, we hypothesize that the alpha- and gamma-synucleins regulate proteasomal function and that beta-synuclein acts as a negative regulator of alpha-synuclein.

English

Natural products have been widely used by human beings. However, sometimes the biological effects of these products are not fully known. Chayotte ( Sechium edule ) is a vegetable used in the folk medicine. Red blood cells (RBC) labeled with technetium-99m (99mTc) have several clinical applications. The aim of this work was to evaluate the influence of an extract of chayotte on the labeling of blood elements with 99mTc using stannous chloride (SnCl 2 ) in the concentrations like to 1.2, 0.006, 0.0005 and 0.0006 μg/ml. The extract of chayote was incubated in various concentrations for 1hour with blood which was withdrawn from Wistar rats. After that SnCl 2 was added and the incubation continued for more 1 h. Elapsed this time 99mTc as sodium pertechnetate (NaTcO 4 ) was toted. The blood was centrifuged and plasma (P) and RBC were isolated, also precipitated with trichloroacetic acid (TCA, 5%) and soluble (S) and insoluble (I) fractions (F) of plasma and cells (C) were determined. The radioactivity (ATI%) was rated in RBC, IF-P and IF-C. The results have showed that extract was able to reduce the radiolabeling using SnCl 2 (0.006, 0.0005 and 0.0006μg/ml). We can speculate that this effect may be on account of the products with oxidant proprieties. Key Words: chayote, red blood cells, plasma proteins, technetium-99m, radiopharmacy. African Journal of Biotechnology Vol.3(9) 2004: 484-488

Ginkgo biloba Extract: Experimental Model to Evaluate its Action on the Labeling of Blood Elements with Technetium-99m and on the Morphometry of Red Blood Cells

We have evaluated the influence of the Ginkgo biloba extract (infusion and crude extract) on the labeling of red blood cells (RBC) and plasma proteins with technetium-99m (Tc-99m). We also studied the morphometry of the RBC treated with Ginkgo biloba extract (EGb). Blood was withdrawn and incubated with EGb. Stannous chloride was added and, then, Tc-99m was added. Plasma (P) and RBC were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) separated. The morphology of the RBC also was evaluated under optical microscope and morphometry. The analysis of the results shows that there is a decrease in the radioactivity on RBC and on IF of P and of RBC with the EGb. The study of the morphology of RBC showed important morphological alterations due to treatment with EGb. These observations were confirmed by morphometry. We suggest that the chemical agents presents in the Ginkgo biloba extract or its active metabolites could act, with: (i) a chelating action of the ions stannous/pertechnetate or (ii) by damages induced in plasma membrane; (iii) or by competition of the cited ions to the same bindings sites; or (iv) with possible generation of reactive oxygen species that could oxidize the stannous ion.

Evaluation of the Biological Effects of a Natural Extract of Chayotte (Sechium edule): A Molecular and Cellular Analysis

Natural products have been widely used by human beings. However, sometimes the biological effects of these products are not fully known. It is concerned that many natural medicines may contain potentially toxic ingredients and contaminants such as heavy metals. Red blood cells (RBC) and plasma proteins labeled with technetium-99m (99mTc) have several clinical applications and it has been reported that some natural products are capable of reducing the efficiency of this radio labeling. The aim of this work was to assess the oxidant or antioxidant action of the agents presents in the extract of Sechium edule verifying its effects on the labeling of blood elements with 99mTc and on the inhibition of the activity of acetyl cholinesterase (AchE). A freshly extract of Sechium edule was administered to Wistar rats during 15 and 60 days. After that, samples (0.5ml) of blood were incubated with stannous chloride (SnCl ) and 99mTc. The 2 blood was centrifuged and plasma (P) and RBC were isolated. P and RBC were also precipitated with trichloroacetic acid and soluble (S) and insoluble (I) fraction (F) were determined. The results have shown that the referred extract was able to reduce the radio labeling in BC to 15 days of treatment (from 98.77%± 0.45 to 90.35%± 5.04) to 60 days of treatment (from 98.77%± 0.45 to 53.53%± 0.91), in IF-BC (from 90.36%± 2.34 to 21.20%± 2.36) and in IF-P to 15 days of treatment (77.20%± 2.19 to 53.26%± 6.69) to 60 days of treatment (77.20%± 2.19 to 11.01%±3.22). The analysis of toxicology was performed by the determination of acetyl cholinesterase activity. In the light of the results the referred extract has not shown inhibitory action due to the activity of the enzyme. In the light of the results obtained we suggest that the referred extract may induce the generation of activity metabolites with oxidant properties with direct action on the labeling process.

The Effect of a Chayotte (Sechium edule) Extracts (Decoct and Macerated) on the Labeling of Blood Elements with Technetium- 9m and on the Biodistribution of the Radiopharmaceutical Sodium Pertechnetate in Mice: an In vitro and In vivo Analysis

The biodistribution of radiopharmaceutical used in diagnostic imaging can be altered by a wide variety of factors. If unknown, the drug interaction with radiopharmaceuticals can lead to misdiagnosis or the necessity to repeat the examination, increasing the dose to the patient. The constituents of herbal products can cause adverse effects. Although natural products are widely used as food or as medicines for humans they can alter the labeling of blood constituents with technetium-99m (99mTc) as wells as the bioavailability of the radiopharmaceutical sodium pertechnetate (99mTcO Na). 99mTc is one of most used radio nuclide 4 in nuclear medicine and in basic research. Some authors described that natural and synthetic drugs may alter the process of radio labeling. Sechium edule (chayotte) is a vegetable very spent in the human nutrition as the popular medicine due its properties such as the diuretic and the hypotensor effects. We evaluated the influence of a chayotte (Sechium edule) extracts (decoct and macerated) on the radio labeling of bloo d elements and on the bioavailability of 99mTcO Na. In this study, blood was withdraw from Wistar rats and 4 the aliquots of blood were incubated with the chayotte extracts decoct and macerated, 0.1g/mL) during 1 hour. After that, blood was incubated with stannous chloride (SnCl ) for 1 hour together with the adding of 2 99mTc in each breaks of 15min until 1 of incubation with SnCl . In each 10 min an aliquot of blood was taken 2 and then plasma (P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid (TCA 5%) and soluble (SF) and insoluble fractions (IF) were separated. For the biodistribution analysis, the 99mTcO Na (0.3mL) was administrated into male Wistar rats which had drunk or not the extract (macerated) 4 for 60 days. After 10 min, the animals were sacrificed, the organs were isolated, the radioactivity determined in a well counter and the percentages of radioactivity per gram (%ATI/g) in the organs was calculated. For the radio images analysis, the animals were treated with the extracts (macerated and decoct) during 15 days, after this period of time, it was administrated doses of 100µCi of 99mTcO Na in the ocular plexus of the 4 animals. The images were obtained after 10min. The qualitative analysis of the images was done. The analysis of the results has demonstrated that the extracts have increased the labeling of blood elements with 99mTc which was incubated together with SnCl , in the times 0 and 30 min to decoct extract (time 0 min: 2 labeling in the C: from 37.97 ± 0.31 to 59.70 ± 0.56; and in the FIC: from 61.76 ± 0.75 to 73.15 ± 0.37; time 30min: labeling in the C: from 84.06 ± 0.91 to 94.75 ± 0.87; and in the FIC: from 76.72 ± 0.26 to 85.37 ± 0.67) and in the times 15 and 30 min to macerated extract (time 15min: labeling in the C: from 29.04 ± 0.31 to 50.13 ± 0.82; time 30min: labeling in the C: from 75.39 ± 0.77 to 83.42 ± 0.51). The %ATI/g was altered in the thyroid (from 4.57 ± 0.58 to 2.03 ± 1.33); lung (from 0.84 ± 0.25 to 0.21 ± 0.02); stomach (from 5.19 ± 1.34 to 2.03 ± 0.60) and blood (from 50.07 ± 3.36 to 32.25 ± 2.32). In the qualitative analysis it was observed an decreased in the performance of the images related to the stomach region. Due to the biodistribution it was related that an extract of eggplant was capable of altering the bioavailability of 99mTcO Na different of a 4 cauliflower extract which has not been able to alter it. It is possible to suggest that some components of chayotte extracts present an oxidant power able to alter the biodistribution of 99mTcO Na, as a tip, we 4 speculate that the referred extract metabolized in the liver may induce the generation of reactive metabolites with oxidant properties, this fact could justify the alteration of the uptake in the organs.

Evaluation of the phytic acid effect on the labeling of blood elements with technetium-99m and on the survival of a strain of Escherichia coli treated with stannous fluoride.

The labeling of red blood cells with technetium-99m (99mTc) depends on a reducing agent and stannous ions, as chloride or fluoride, are widely utilized. This labeling may also be altered by drugs. Moreover, some authors have reported that the survival of Escherichia coli (E. coli) cultures decreases in presence of stannous ions. Phytic acid is present in the daily diet and we evaluated its influence on: (i) the labeling of blood elements with 99mTc and (ii) on the survival of an E. coli strain treated with stannous fluoride. Heparinized whole blood was withdrawn from Wistar rats and it was incubated with stannous chloride and with 99mTc, as sodium pertechnetate, centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P and BC were also precipitated with trichloroacetic acid, centrifuged and soluble (SF) and insoluble fractions (IF) isolated. E. coli culture was treated with stannous fluoride in presence of phytic acid. As phytic acid altered the fixation of 99mTc on BC, on IF-P and on IF-BC and, moreover, it abolished the lethal effect of stannous fluoride on the E. coli culture, we can suggest that, probably, phytic acid would have chelating properties to the stannous ions.

Effect of Ginkgo biloba on the labeling of blood elements with technetium-99m: in vitro study

Ginkgo biloba is the phytoterapic most used in popular medicine in the treatment of cerebral senescence. Red blood cells (RBC) labeled with technetium-99m (Tc-99m) is used for several evaluations in nuclear medicine. This labeling depends on a reducing agent, usually the stannous ion. Any drug, which alters the labeling of the tracer, could be expected to modify the disposition of the radiopharmaceutical. We have evaluated the influence of the Ginkgo biloba extract on the labeling of RBC and plasma proteins with Tc-99m. Blood was withdrawn and incubated with Ginkgo biloba extract (0; 0.004; 0.04; 0.4; 4; 20 and 40 mg/ml). Stannous chloride (1.2 ml/ml) was added and, then, Tc-99m was added. Plasma (P) and blood cells (RBC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) separated. The analysis of the results shows that there is a decrease in the radioactivity (from 97.7 ± 0.7 to 49.5 ± 3.9%) in RBC with the drug (4 mg/ml). In the labeling process of RBC with Tc-99m, the stannous and pertechnetate ions pass though the membrane, so, we suggest that the Ginkgo biloba effect can be explained by (i) an inhibition of the transport of these ions, (ii) damage in membrane, (iii) competition with the cited ions for the same binding sites, or (iv) possible generation of reactive oxygen species that could oxidize the stannous ion.

Effect of ginkgo biloba on the in vitro labeling of red blood cells and plasma proteins with technetium‐99M

AbstractGinkgo biloba is the phytoterapic most used in popular medicine in the treatment of inflammation, free‐radical with traumatic brain injury and Alzheimer's dementia. Red blood cells (RBC) labeled with technetium‐99m (Tc‐99m) are used for several evaluations in nuclear medicine. This labeling depends on a reducing agent, usually the stannous ion. Any drug which alters the labeling of the tracer could be expected to modify the disposition of the radiopharmaceutical. We have evaluated the influence of the Ginkgo biloba extract on the labeling of RBC and plasma proteins with Tc‐99m. Blood was withdrawn and incubated with Ginkgo biloba extract (0; 0.004; 0.04; 0.4; 4 and 20 mg/mL). Stannous chloride (1.2 μl/mL) was added and, then, Tc‐99m was added. Plasma (P) and blood cells (RBC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) separated. The analysis of the results shows that there is a decrease in the radioactivity (from 97.7 ± 0.7 to 49.5 ± 3.9%) in RBC with the drug (4mg/mL). In the labeling process of RBC with Tc‐99m, the stannous and pertechnetate ions pass though the membrane, so, we suggest that the Ginkgo biloba effect can be explained by (i) an inhibition of the transport of these ions, (ii) damage in membrane, (iii) competition with the cited ions for the same binding sites, or (iv) possible generation of reactive oxygen species that could oxidize the stannous ion.

Intestinal absorbability of wheat allergens, subunits of a wheat alpha-amylase inhibitor, expressed by bacteria.

Wheat CM2, CM3 and CM16 proteins are known as subunits of the tetrameric alpha-amylase inhibitor as well as major allergens to baker's asthma. The purpose of this study is to produce these CM proteins by bacteria in a quantity adequate for studying the penetration characteristics of the CM proteins through intestinal mucosa in rats and Caco-2 cells. cDNAs encoding the mature proteins were expressed in Escherichia coli and purified by an Ni2+-chelating column. The recombinant proteins were radioiodinated and admministered orally to rats or applied to the apical site of the Caco-2 cell monolayer. The radioactivity in the trichloroacetic acid-insoluble fraction, which was mainly composed of peptides with molecular mass less than that of the intact CM proteins, in the serum and the basolateral medium was highest in recombinant CM3. Accordingly, the intestinal absorption of these three proteins in the form present in wheat should be evaluated.

The effects of tgf- ; IGF, insulin-like growth factor ; DTT, dithiothreitol. EDTA, ethylenediaminetetraacetic acid ; PMSF, phenylmethylsulfonyl fluoride ; TBS, tris buffer saline. SDS-PAGE, sodium dodecyl sulfate-polyacrilamide gel electrophoresis.1 on chick embryo retina development in vitro

This paper studies the effect exerted by TGF-β1 on the development of chick embryo retina cultured in vitro. The addition of TGF-β1 to retinal explants inhibited DNA synthesis, measured as 3H-thymidine incorporation into trichloroacetic acid-insoluble fraction, while it increased both wet weight and protein content, in particular that of extracellular matrix proteins. Lastly, in explants treated with TGF-β1 an increment in the level of fibronectin was demonstrated by means of Western blotting analysis.

Isolation of mammalian pol beta type DNA polymerase in shoot tips of germinated seedlings of IR-8 rice (Oryza sativa L.).

A single polypeptide of approx. 67 kDa mol.wt. with DNA polymerase activity has been chromatographically purified from shoot tips of 72 hr. grown germinated seedlings of rice (Oryza sativa.L.cv IR-8). An approx. 4800 fold enrichment of specific activity was measured by the incorporation of 3H-dTMP into trichloroacetic acid insoluble fraction, using activated calf thymus DNA as template-primer. The enzyme uses different types of DNA but not RNA as a template. The enzyme requires Mg+2, high KCl, and is highly sensitive to dideoxyribonucleoside triphosphate but is unaffected by aphidicolin, suggesting a plant counterpart of mammalian DNA polymerase beta. Replication of M13mp18 single stranded DNA by extension of 5'32P-labeled 17-mer primer(-40) showed distributive type of DNA synthesis by the rice DNA polymerase beta, which is a characteristic feature of mammalian DNA polymerase beta.

Bacterioplankton cell growth and macromolecular synthesis in seawater cultures during the North Atlantic Spring Phytoplankton Bloom, May, 1989

We performed a series of seawater culture experiments on surface mixed layer samples during the spring phytoplankton bloom in the North Atlantic Ocean. Diluted (20% unfiltered + 80% 0.22 μm filtered) and untreated "whole" seawater samples were incubated up to 40 hour and sampled periodically for cell numbers, biovolume, and incorporation of (3)H-thymidine and -leucine. Abundance and biovolume increased exponentially at similar rates in diluted and whole samples, suggesting that removal by bacteriovores was low compared with growth. The exponential increase in biovolume was due to increases in cell numbers and mean cell volume. Generation times (i.e., 0.693/μ) averaged 36-53 hour in these surface (10 m) samples. Ninety percent of the tritiated thymidine incorporation (TTI) into cold trichloroacetic acid-insoluble cell fractions was recovered after extraction with NaOH and phenolchloroform, indicating that catabolism of thymidine and its appearance in RNA or protein was very low. The percentage of thymidine recovered in DNA did not change over the 40 hour of incubation and was the same as in water column samples. Rates of thymidine and leucine incorporation also increased exponentially. Incorporation rates tended to increase more rapidly than cell numbers or biovolume, though the differences were not significantly different, due to the small number of samples and variability over the time courses. Differential rates of increase in cellular properties during growth might indicate a lack of coupling between incorporation and production over time scales of hours-days. This in turn may reflect unbalanced growth of bacterial assemblages, which is an adaptation to variable conditions in the upper ocean in this season. Nonequality of rate constants for cells and incorporation yields conversion factors that are either higher or lower than would be calculated from balanced growth (i.e., rates of increase in numbers and incorporation rates equal), depending on the calculation approach chosen. An alternative approach to calculating conversion factors (the modified derivative approach) is proposed, which is insensitive to differential rates of increase of abundance and incorporation.

Identification of insulin in chick embryo retina during development and its inhibitory effect on DNA synthesis.

Incubation of chick embryo retinal explants with insulin resulted in a pronounced inhibition of thymidine uptake and incorporation into trichloroacetic acid-insoluble fraction. The inhibitory effect was highest with explants from embryos at day 7 and day 8, and thereafter it declined markedly with the age of embryos until day 11. A time-course study of the effect revealed that the inhibition occurred after a lag time; both thymidine uptake and incorporation were not altered significantly after 2-6 h of incubation with insulin, but began to decrease thereafter, reaching the maximum after 16 h. The effect was also dose dependent. After 16 h of incubation, the maximal inhibition (65%) was found with 10(-8) M insulin. Insulin caused similar effects also on thymidine kinase activity. All these effects were obtained by using minimal essential medium without glutamine. The addition of glutamine to the medium reduced the inhibitory effect of insulin. Retinas of chick embryos contain immunoreactive insulin. Retinal immunoreactive insulin was at the highest level (1.12 ng/mg of protein) in the youngest retinas studied (day 6), then it declined with age, reaching the lowest value (0.58 ng/mg of protein) at day 14. This value did not vary significantly during the third week of development. A potential biological role of insulin in retinal development is discussed.

Uptake, disposition and depuration of domoic acid by blue mussels ( Mytilus edulis)

Domoic acid (DOM), the toxin involved in amnesic shellfish poisoning, was presented to live mussels (at 5°C, 28 0 00 ) in dissolved form (125 nM) and as food encapsulated in liposomes. Less than 1% of dissolved DOM and up to 6% of food-borne DOM was incorporated into mussel tissues. DOM absorbed from solution was most concentrated in gills and kidney, whereas DOM ingested as food was most concentrated in digestive gland and kidney. Gonad, muscle, foot and connective tissues retained the lowest concentrations of toxin. Compared to their proportion of body weight; kidney, digestive gland and gill retained larger than expected proportions of total toxin burden. The concentration of toxin in mussel tissues did not decrease consistently over a depuration period of 48 h, nor did DOM appear to be translocated to any tissue for storage. Small amounts of DOM were eliminated in faeces and larger amounts in dissolved form. Over 80% of intracellular DOM was associated with the TCA (trichloroacetic acid) soluble fraction of digestive gland tissue. DOM in the TCA insoluble fraction was positively correlated with overall DOM concentration.

9-Hydroxybenfiuron

Primary screening in vitro and study on the mode of action of 9-hydroxybenfluron (HBF) in both murine P388 leukemia and Ehrlich ascites carcinoma cells have been performed. Metabolite HBF is approximately twice as effective as a reference drug (benfluron). To elucidate the biochemical mode of action, the effect of HBF on the biosynthesis of macromolecules indicated by the incorporation rate of [14C]adenine (in DNA and RNA), [14C]thymidine (in DNA), [14C]uridine (in RNA) and [14C]valine (in protein) was studied in concentration and time dependence. HBF inhibited incorporation of all four precursors into the trichloroacetic acid-insoluble fraction of Ehrlich ascites cells. The fact that incorporation of these four precursors is inhibited suggests that the effect of HBF lies at an underlying level of energy generation or transfer rather than at specific reactions in the biosynthesis of DNA and proteins.