High Performance Liquid Chromatography of Molecular Species from Free Sterols and Sterylglycosides Isolated from Oat Leaves and Seeds

Free sterols and sterylglycosides (SG) from oat leaves and seedswere isolated by conventional thin layer chromatography (TLC)and subjected to high performance liquid chromatography (HPLC)for resolution of molecular species. Acylsterylglycosides, isolatedby TLC, were converted to SG by mild alkaline hydrolysis anddetermined as SG. Sterols and SG were injected onto the columnwithout any chemical treatment and the separated species weredetected at 200 nm. The separation of SG-species follows exactlythe separation of free sterols. Though gas liquid chromatography still is the method of choice,advantages of HPLC is to analyse directly the SG-species withouthydrolysis and derivatization as compared to GLC. After TLCthe sterol- and the SG-fraction are injected directly onto thecolumn. This is extremely important for labile sterylglycosidesor sterols, as demonstrated for the avenasterols. ¹ Preliminary reports have been presented on the "4. Arbeitstagung,Pflanzliche Lipide", October 7–8, 1983 in M?nster (FRG)and on the "6th International Symposium on the Structure, Functionand Metabolism of Plant Lipids", Neuchatel, Switzerland, July16–20, 1984. (Received November 12, 1984; Accepted January 14, 1985)     

Ascaris suum: protective immunity in pigs immunized with products from eggs and larvae

Parasite products were collected at three distinct phases of development of Ascaris suum, and their immunogenicity was determined after injection into rabbits and pigs. Products were derived from (1) the hatching fluid of infective eggs; (2) the conditioned medium of 2nd-stage larvae that developed to 3rd stage in vitro in defined medium; and (3) the conditioned medium of 3rd-stage larvae that developed to 4th stage in vitro in defined medium. Protein profiles from these three preparations, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were less complex than that of extracts from homogenized A. suum larvae. Hyperimmune rabbit antiserum raised against either egg products, 2nd- to 3rd-stage larval excretory-secretory products, or 3rd- to 4th-stage larval excretory-secretory products showed strong homologous reactions after immunoelectrophoresis, but relatively weak cross-reactions with the other preparations. A combined enteral immunization of pigs with egg products and parenteral immunization with the 2nd- to 3rd-stage larval excretory-secretory products, and 3rd- to 4th-stage larval excretory-secretory products induced antibody to each preparation and significant protective immunity to a challenge exposure with 10,000 A. suum eggs. However, a marked pathological response to larvae migrating in the liver after challenge exposure was also induced.     

The innervation of the pyloric region of the crab,Cancer borealis: Homologous muscles in decapod species are differently innervated

Summary The muscles of the pyloric region of the stomach of the crab,Cancer borealis, are innervated by motorneurons found in the stomatogastric ganglion (STG). Electrophysiological recording and stimulating techniques were used to study the detailed pattern of innervation of the pyloric region muscles. Although there are two Pyloric Dilator (PD) motorneurons in lobsters, previous work reported four PD motorneurons in the crab STG (Dando et al. 1974; Hermann 1979a, b). We now find that only two of the crab PD neurons innervate muscles homologous to those innervated by the PD neurons in the lobster,Panulirus interrruptus. The remaining two PD neurons innervate muscles that are innervated by pyloric (PY) neurons inP. interruptus. The innervation patterns of the Lateral Pyloric (LP), Ventricular Dilator (VD), Inferior Cardiac (IC), and PY neurons were also determined and compared with those previously reported in lobsters. Responses of the muscles of the pyloric region to the neurotransmitters, acetylcholine (ACh) and glutamate, were determined by application of exogenous cholinergic agonists and glutamate. The effect of the cholinergic antagonist, curare, on the amplitude of the excitatory junctional potentials (EJPs) evoked by stimulation of the pyloric motor nerves was measured. These experiments suggest that the differences in innervation pattern of the pyloric muscles seen in crab and lobsters are also associated with a change in the neurotransmitter active on these muscles. Possible implications of these findings for phylogenetic relations of decapod crustaceans and for the evolution of neural circuits are discussed.Abbreviations ACh acetylcholine - Carb carbamylcholine - cpv muscles of the cardio-pyloric valve - cpv7n nerve innervating muscle cpv7 - cv muscles of the ventral cardiac ossicles - cv1n nerve innervating muscle cvl - cv2n nerve innervating muscle cv2 - EJP excitatory junctional potential - IC inferior cardiac neuron - IV inferior ventricular neuron - IVN inferior ventricular nerve - LP lateral pyloric neuron - LPG lateral posterior gastric neuron - lvn lateral ventricular nerve - mvn medial ventricular nerve - p muscles of the pylorus - PD pyloric dilator neuron - PD _in intrinsic PD neuron - PD _ex extrinsic PD neuron - pdn pyloric dilator nerve - PY pyloric neuron - pyn pyloric nerve - STG stomatogastric ganglion - VD ventricular dilator neuron     

Factors contributing to the in vitro development of Ascaris suum from second-stage larvae to mature adults

When a three-step roller culture system was used, second-stage larvae of Ascaris suum, artificially hatched from eggs, developed in high numbers to the fourth stage, and a few to young and mature adults. The culture system consisted of (1) Medium KW-2 supplemented with 10 mM L-cysteine for the first 4 days, and with 5 mM L-cysteine for the following 7 days; (2) followed by Medium API-18 for 7 days; and (3) thereafter, by Medium API-1 supplemented with hemin (bovine) at a concentration of 24 micrograms/m1. Cultures were gassed with 95% nitrogen-5% carbon dioxide for the first 4 days and 85% nitrogen-5% oxygen-10% carbon dioxide thereafter, and incubated at 39 C. Two mature females that produced unfertilized eggs and a mature male with spermatozoa were the most advanced stages attained. The mature females were obtained in 67 and 73 days; and the largest female measured 110 mm. The latter produced 1,356,000 unfertilized eggs, from days 67 to 125. The mature male was obtained in 80 days; it measured 77 mm long and had paired spicules that were 1.5 mm long. Development of A. suum in three other culture systems showed that deletion of Medium API-18 or its substitution by Medium KW-2 limited development to late fourth stage and early, young adults, respectively; and the use of Medium API-1 without hemin limited development to early fourth stage.     

Independent immunodominant and immunorecessive tumor-specific antigens on a malignant tumor: antigenic dissection with cytolytic T cell clones

We have analyzed the complexity of a unique tumor-specific transplantation antigen expressed by the murine ultraviolet light-induced fibrosarcoma 1591-RE. This tumor is highly immunogenic and is regularly rejected by normal mice. We have derived a cloned cytolytic T cell line showing a reactivity pattern representative of the cytolytic response of the host rejecting this regressor tumor. Using this T cell line (anti-A), variants of 1591-RE (1591-A-) were selected in vitro that had lost the same antigen as progressor variants of 1591-RE selected by the host in vivo. The in vitro derived variant was then used to generate a second T cell clone (anti-B) that recognized an antigen on the parental tumor that had been retained by the variants derived in vitro. Host-selected progressor variants were also found to have retained this antigen. By selecting for variants in vitro from the parental tumor with the anti-B T cell line, it was shown that the two different antigens (A and B) present on the parental tumor were lost independently of each other. Despite the independence of these two antigens, the host T cell response to the parental regressor tumor was invariably restricted to only the "immunodominant" A antigen.     

A simple high capacity freeze-drier for histochemical use

Summary A new freeze-drier for histochemical use is described. It uses a refrigerated cooling bath for outer cooling and large amounts of phosphorous pentoxide as water vapor trap.The main features are a very high drying capacity, a simple, reliable easy-to-handle construction and a series of safety devices which, including a rigid stainless steel vacuum chamber which cannot implode, ensures reproducable results.Estimations of relative dryness can be performed during drying. An extra blind flange entrance to the vacuum chamber and the use of standard vacuum connections makes the apparatus versatile. Thus it can be used also for chemical freeze-drying.The apparatus was developed for use with the Falck-Hillarp fluorescence technique for histochemical visualization of monoamines. It gives excellent results with this technique both with peripheral tissues and brain tissue. As many as 20–25 whole brains from adult rats can be processed simultaneously within 3 days.     

A new member of a secretory protein gene family in the dipteranChironomus tentans has a variant repeat structure

Summary We describe the structure of a gene expressed in the salivary gland cells of the dipteranChironomus tentans and show that it encodes 1 of the approximately 15 secretory proteins exported by the gland cells. This sp115,140 gene consists of approximately 65 copies of a 42-bp sequence in a central uninterrupted core block, surrounded by short nonrepetitive regions. The repeats within the gene are highly similar to each other, but divergent repeats are present in a pattern which suggests that the repeat structure has been remodeled during evolution. The 42-bp repeat in the gene is a simple variant of the more complex repeat unit present in the Balbiani ring genes, encoding four of the other secretory proteins. The structure of the sp115,140 gene suggests that related repeat structures have evolved from a common origin and resulted in the set of genes whose secretory proteins interact in the assembly of the secreted protein fibers.     

Aluminum geochemistry in peatland waters

The chemical speciation of aluminum was examined in surface water samples from Sphagnum peatlands in north-central Minnesota, from peatlands along the Canadian east coast, and from bogs in the Pennine Mountain area of England. In highly organic ([DOC] 50 mg L^–1 ), low pH waters, 80–90% of total dissolved Al was complexed with organic matter (OM), while in waters with low DOC ([DOC] 5 mg L^–1) 54–86% of total dissolved Al existed as Al⁺³ or other inorganic Al species. Batch titrations of OM with Al revealed a high Al binding capacity, 1.4–2.8 mol (mg DOC)^–1, that generally was unsaturated with Al. Titrations of OM with Al in conjunction with a continuous distribution model were used to determine Al-OM conditional stability constants. Binding capacity (mol Al (mg DOC)^–1) and strength (formation constant) increased from pH 3 to 5 but decreased above pH 5 due to formation of AI-hydroxy species including A1(OH)₃ (s). The high binding capacity of OM in bog waters facilitates metal mobility, especially in low pH (< 5) wetlands where metal solubility is high and OM concentrations are highest. Results showed that the relative degree of organic matter saturation with metal ions was important in modeling AI speciation in bog waters.     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.