A tissue culture model for studying ethanol toxicity on embryonic heart cells

A tissue system in which fibroblasts and myocytes from chick embryonic hearts were separately maintained was used to study the toxicity of ethanol. To reproduce the teratogenic effects of acute, high concentrations of ethanol typical of binge drinking, an open tissue culture system was employed. With open cultures, the cells were initially exposed to peak alcohol levels for approximately 6 hr and were exposed to decreasing concentrations of ethanol for the remainder of each 24 hr period. After the first day of ethanol exposure, there was substantial cell loss in both fibroblast and myocyte cultures. Alcohol-induced cell loss was dose-dependent. Despite decreased cell density after the first day of ethanol exposure, the surviving cells differentiated into monolayers of fibroblasts or beating cardiac muscle fibers. However, both ethanol-exposed fibroblasts and myocytes appeared atrophic, that is, smaller and shrunken. Electrophoretic analysis or these ethanol-exposed fibroblast and myocyte cultures revealed specific reduction in the cellular contents of -actinin, myosin, and actin. These decreases in cytoskeletal proteins may be responsible for the morphological abnormalities noted in these cells.Abbreviations FAS, fetal alcohol syndrome - NAD, nicotinamide adenine dinucleotide - PAS, periodic acid Schiff - SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Conformationally constrained opioid peptide analogs with novel activity profiles

Novel conformationally constrained opioid peptide analogs with antagonist, mixed agonist/ antagonist or agonist properties were developed. TIP(P)-related antagonists showed unprecedented antagonist potency and receptor selectivity, and may have potential for use in analgesia in combination with agonists. A definitive model of their receptor-bound conformation was developed. Three prototype mixed agonist/ antagonists were discovered. They represent the only known compounds with this pharmacological profile and, as expected, one of them was shown to be a potent analgesic and to produce no dependence and less tolerance than morphine. Novel dipeptide derivatives turned out to be potent and selective agonists. Because of their low molecular weight and lipophilic character, these compounds may cross the blood-brain barrier and, thus, may have potential as centrally acting analgesics.     

Evidence suggesting energy-dependent formaldehyde transport in an RuMP-type methylotroph (T15)

Formaldehyde accumulation ratios ([¹⁴CH₂O]_i/[¹⁴CH₂O]_o) as high as 12-fold were measured in anaerobic, CH₃OH-energized, whole cell suspensions of the ribulose monophosphate (RuMP)-type methylotrophic strain T15. Uptake kinetics were extremely rapid, enabling the attainment of equilibrium in only 10–30 s. Transport appears to be energy-dependent and associated with the protonmotive force (pmf). Anaerobic incubation with 5 M carbonyl p-(trifluoromethoxy)-phenylhydrazone (FCCP) led to 70%–90% reduction of the accumulation ratio. Though not as pronounced, diminished uptake was also observed in the presence of 140 M nigericin, 161 M valinomycin and 90 mM KSCN, commensurate with their effects on pmf. Accumulation of CH₂O as a function of external pH followed a trend more similar to that of pmf than either pH or . Preventing energization by incubation with 100 M N,N-dicyclohexylcarbodiimide (DCCD) led to nearly 80% inhibition of CH₂O transport. Over short time periods it was possible to chase accumulated ¹⁴CH₂O from previously loaded cells by collapsing pmf; however, this technique also indicated that significant ¹⁴CH₂O incorporation began to occur within 3 min.Abbreviations FCCP Carbonyl cyanide p-(trifluoromethyoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - RuMP ribulose monophosphate - TPP⁺ tetra[U-¹⁴C]phenylphosphonium - pmf protonmotive force     

Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

Unexpected Transformations of Natural Chlorins under Treatment with Dichlorodicyanobenzoquinone

The treatment of natural chlorins with 2,3-dichloro-5,6-dicyanobenzoquinone resulted not only in the intramolecular cyclization of the propionic acid residue in position 17 with the formation of an additional -lactone cycle at the pyrrole ring D, but also in the oxygen-assisted oxidation of 8-ethyl group in ring B to an -methoxyethyl substituent.     

δ-Toxin,unlike melittin,has only hemolytic activity and no antimicrobial activity: Rationalization of this specific biological activity

The antimicrobial activity of a synthetic peptide corresponding to -hemolysin had been examined. The peptide didnot exhibit antimicrobial activity against gram negative and gram positive micro-organisms unlike other hemolytic peptides like melittin. This lack of antibacterial activity arises due to the inability of -hemolysin to perturb the negatively charged bacterial cell surface and permeabilize the bacterial plasma membrane. However, the red blood cell surface has a structure considerably different from bacteria, and does not act as a barrier to molecules reaching the lipid membrane. Hence -toxin can lyse erythrocytes. Thus, the specificity in biological activity has been rationalized in terms of differences, in the interaction of the toxin with the bacterial and red blood cell surfaces.     

Influence of field bed position,ground surface color,mycorrhizal fungi,and high root-zone temperature in woody plant container production

High root-zone temperatures can stress plants and reduce nursery productivity of container produced crops. Field studies were conducted to study position of containers in field beds, ground surface color, mycorrhizal fungi and high root-zone temperatures in the production of selected woody plants. Root-zone temperature profiles in containers were established to determine nursery production conditions for white and black ground bed surfaces. White surfaces increased container medium temperatures in beds of plants with open canopies by 2–4°C compared to full canopied plants. Under field conditions with container medium temperatures as high as 40–50°C, the open canopiedBerberis thunbergii DC. Atropurpurea,Pinus eldarica Medw. andBuxus microphylla Seibold and Zucc. were more susceptible to temeprature stress compared to the more close canopiedPittosporum tobira (Thunb.) Ait. Wheeler's dwarf. When compared to controls,P. tobira colonized with mycorrhizal fungi [Glomus etunicatus Baker and Gerd. andGlomus fasciculatum (Thax.sensu Gerd.) Gerd. and Trappe] had increased shoot growth in all bed areas except the western exposure, and increased root growth in western and eastern bed regions. Greatest root damage generally occurred in containers of colonized and noncolonizedB. thunbergii in southern and western bed exposures. Mycorrhizal colonization did not improve plant growth of the more high temperature susceptibleB. thunbergii.     

Variation in stable isotope signatures of seston and a zooplanktivorous fish in a eutrophic Chinese lake

Temporal and spatial changes in ¹³C and ¹⁵N of seston (mainly phytoplankton) and isotopic relationship between seston and the lake anchovy (Coilia ectenes) were studied in the large eutrophic freshwater Lake Chaohu in China. Much of the spatial and temporal variation in ¹³C of lake anchovies was explained by variation in seston, indicating a strong link between pelagic primary production and higher order consumers. Because the lake is shallow, there were no significant differences in ¹³C and ¹⁵N of seston between surface and overlying waters. Spatially, the relatively high ¹³C and ¹⁵N of seston in the western part of the lake might be due to high levels of anthropogenically derived N and C introduced from the surrounding cities through sewage drainage systems. The trophic position of the lake anchovy in the food web of Lake Chaohu was estimated to be 2.9–4.1 (3.5 ± 0.4), which agrees well with the previous stomach content analysis suggesting that the lake anchovy fed both on zooplankton and small planktivorous fishes.     

Effect of delta-lysin on the phase transitions of lipid assemblies

X-ray small-angle diffraction, differential scanning calorimetry (DSC), and temperature scanning densitometry (TSD) were used to study the effect of -lysin on the phase transitions of lipid assemblies from 1,2-0-dixehadecyl-sn-glycero-3-phosphoholine (DHPC). The experiments were carried out in excess of water in a temperature range of 0–55 °C, and at low peptide concentrations between 10^-4 and 10^-2 moles peptide per mole phospholipid. The incorporation of -lysin into lipid assemblies alters the lipid structure without significant changes on the temperatures of phase transition from gel to liquid crystalline phase. The temperature of the main transition was nearly unaffected. A reduction in the transition volume of the lipids with increasing concentrations of -lysin was observed. The minor changes in these parameters were interpreted as long-range structural changes caused by the peptide incorporation. The results are discussed in terms of the concept of cooperative phase transition of entire clusters occurring within a membrane implying that relative stable domains of gel phase, and liquid crystalline phase co-exist.     

Histological distribution and developmental changes of tropomyosin isoforms in three chicken digestive organs

Summary Histological localization of tropomyosin isoforms in three digestive organs from embryonic and adult chickens was performed by using rabbit antisera against chicken skeletal muscle tropomyosin and against low-Mr-type tropomyosin from chicken small intestine mucosa. The former antiserum (named TM-SH) reacted with , , and high-Mr-type isoforms, and the latter (named TM-HL) reacted with , , high-Mr-type and low-Mr-type isoforms. and Isoforms were detected in muscle cells of the muscular layer and the muscularis mucosa. Low-Mr-type isoforms, however, were detected along the cell membrane and cytoplasm of almost all nonmuscle cells, especially in terminal webs of epithelial cells. Developmental changes of tropomyosin isoforms in digestive organs were studied by two-dimensional gel electrophoresis and image analysis. The relative amounts of and isoforms increased in the course of development, but those of low-Mr-type and high-Mr-type isoforms decreased.     

A rapid and simple method for staining of the crystal protein ofBacillus thuringiensis

Summary A rapid and simple method of staining for the crystal protein (-endotoxin or parasporal body) ofBacillus thuringiensis has been developed. Changes in colonial morphology were observed when cells lost their ability to form crystal protein or both crystal protein and spore.     

Pod development of groundnut (Arachis hypogaea L.) in solution culture

Normal pods (containing seed) of groundnut (Arachis hypogaea L.) (cv. TMV-2) were successfully raised in darkened, aerated, nutrient solution, but not in the light. The onset of podding was evident 7 to 8 d after gynophores were submerged in the darkened nutrient solution. An examination of pods and submerged portions of gynophore surfaces by scanning electron microscopy showed the presence of two distinctly different protuberances: unicellular root-hair-like structures that first developed from epidermal cells of the gynophores and developing pods; and branched septate hairs that developed later from cells below the epidermal layer. The septate hairs became visible only after the epidermal and associated unicellular structures had been shed by the expanding gynophore and pods. Omission of Mn and Mg from the podding environment increased pod and seed weight, whilst omission of Zn reduced pod and seed weight.     

A model for cleavage ofO-glycosidic bonds in glycoproteins

The present work investigated the possibility of cleavage of -linkages between mannose or galactose and serine/threonine residues by -mannosidase and -galactosidase. The study was carried out initially with model synthetic compounds imitating theO-glycosidic bond in glycoproteins, and further with glucoamylase. It was shown that -mannosidase and -galactosidase can hydrolyse these linkages after proteolytic digestion of glucosamylase.     

The wise use of wetlands: identifying development options for Ghana's coastal Ramsar sites

The Government of Ghana has recently declared five coastal wetlands to be Ramsar sites. This requires the wise use of wetlands of international importance for bird habitat, but does not preclude human habitation or ecologically friendly development. The sites are Muni-Pomadze, Densu Delta, Sakumo, Songor and Keta. An attempt was made to identify and assess a range of development options for initiation within the sites that would be compatible with the environmental concerns of Ramsar, while also being technically and economically viable and socially acceptable to the communities in the sites. The appraisal included developing a detailed understanding of the physical, biological, human and institutional resources of the areas, the land uses and prevailing livelihood systems of local people, and the pressures placed on the natural resources of the wetlands. Many of the developments nominated by stakeholders were rejected, but twenty topics passed the initial tests. These were considered in more detail using information collected for the study by national specialists. Most of the options were considered suitable for local level implementation by community groups or local entrepreneurs. Government assistance will be needed to ensure an enabling institutional framework for small-scale business development is provided.     

Emission of gaseous nitrogen oxides from an extensively managed grassland in NE Bavaria, Germany.

We analysed the stable isotope composition of emitted N₂O in a one-year field experiment (June 1998 to April 1999) in unfertilized controls, and after adding nitrogen by applying slurry or mineral N (calcium ammonium nitrate). Emitted N₂O was analysed every 2–4 weeks, with additional daily sampling for 10 days after each fertilizer application. In supplementary soil incubations, the isotopic composition of N₂O was measured under defined conditions, favouring either denitrification or nitrification. Soil incubated for 48 h under conditions favouring nitrification emitted very little N₂O (0.024 mol g_dw ^–1) and still produced N₂O from denitrification. Under denitrifying incubation conditions, much more N₂O was formed (0.91 mol g_dw ^–1 after 48 h). The isotope ratios of N₂O emitted from denitrification stabilized at ¹⁵N = –40.8 ± 5.7 and ¹⁸O = 2.7 ± 6.3. In the field experiment, the N₂O isotope data showed no clear seasonal trends or treatment effects. Annual means weighted by time and emission rate were ¹⁵N = –8.6 and ¹⁸O = 34.7 after slurry application, ¹⁵N = –4.6 and ¹⁸O = 24.0 after mineral fertilizer application and ¹⁵N = –6.4 and ¹⁸O = 35.6 in the control plots, respectively. So, in all treatments the emitted N₂O was ¹⁵N-depleted compared to ambient air N₂O (¹⁵N = 11.4 ± 11.6, ¹⁸O = 36.9 ± 10.7). Isotope analyses of the emitted N₂O under field conditions per se allowed no unequivocal identification of the main N₂O producing process. However, additional data on soil conditions and from laboratory experiments point to denitrification as the predominant N₂O source. We concluded (1) that the isotope ratios of N₂O emitted from the field soil were not only influenced by the source processes, but also by microbial reduction of N₂O to N₂ and (2) that N₂O emission rates had to exceed 3.4 mol N₂O m^–2 h^–1 to obtain reliable N₂O isotope data.