The role of the structural parts of bacterial lipopolysaccharide in its direct immunosuppressive activity]

The direct immunosuppressive activity of lipopolysaccharides (LPS) and their structural parts (O-chains, R-core, lipid A), obtained from Salmonella, Pseudomonas and Burkholderia, was studied. LPS preparations were extracted by the phenol-water method. Structural parts of LPS were obtained by acetic acid hydrolysis and gel filtration. The study demonstrated that all these preparations, when injected intraperitoneally into mice, did not affect the level of delayed-type hypersensitivity (DTH) to the test antigen in the animals. After redox treatment all LPS preparations became capable of suppressing DTH. After redox treatment such immunosuppressive activity could be observed in lipid A, while O-specific chains and R-core remained inactive. After phenol treatment immunosuppressive activity disappeared. Chemical groups capable of activation were likely to be located in lipid A or in lipid A-associated protein.     

Source of electrons for extracellular Fe(III) reduction in iron-deficient bean roots

Iron-deficient Phaseolus vulgaris L. cv. Prelude developed a high reducing capacity for extracellular Fe(III) at the root surface. This reduction was competitively inhibited by Nitro-Blue Tetrazolium salt (Nitro-BT) which was deposited as a blue precipitate within the epidermis cells of the youngest root parts. Root respiration was not influenced by Nitro-BT. The intracellular reduction of Nitro-BT could largely be prevented by excess extracellular Fe(III)EDTA. Iron-sufficient control plants reduced both extracellular Fe(III)EDTA and intracellular Nitro-BT at a much slower rate. A role of cytosolic NADH or NADPH as direct electron donors for the enhanced Fe(III) reduction at the plasmalemma is indicated. NAD⁺-3-phosphate dehydrogenase activity was higher in preparations from iron-deficient root parts than in preparations from control root parts. Ferricyanide, dichlorophenolindophenol and phenazine methosulfate were also reduced at an increased rate by iron-deficient roots. We conclude that a trans-plasma membrane electron transfer, mediated by a membrane-bound reductase, is responsible for the reduction of extracellular Fe(III).     

Adsorption of a hydrophobic mutagen to five contrasting dietary fiber preparations.

The ability of five plant cell wall (dietary fiber) preparations with contrasting compositions to adsorb in vitro the hydrophobic, environmental mutagen, 1,8-dinitropyrene (DNP), was investigated. Many of the fruits and vegetables in Western diets are from dicotyledonous (broad leaved) plants and the dietary fiber from these consists mainly of unlignified cell walls. A representative of this wall type, prepared from immature cabbage leaves, showed little ability to adsorb DNP. Two other cell-wall preparations, representing lignified walls of dicotyledons and unlignified walls of vegetative parts of grasses and cereals (monocotyledons belonging to the family Poaceae), adsorbed DNP much more effectively. However, two further preparations, representing suberized walls of cork cells and lignified walls of vegetative parts of grasses and cereals, were the most effective in adsorbing DNP. Extrapolation of these data to the in vivo situation would indicate that increased consumption of the vegetative parts of grasses or cereals and plant material containing cork cells, for example potato skins, could be effective in removing hydrophobic mutagens from potential contact with colonic mucosal cells.     

Adsorption of a hydrophobic mutagen to five contrasting dietary fiber preparations

The ability of five plant cell wall (dietary fiber) preparations with contrasting compositions to adsorb in vitro the hydrophobic, environmental mutagen, 1,8-dinitropyrene (DNP), was investigated. Many of the fruits and vegetables in Western diets are from dicotyledonous (broad leaved) plants and the dietary fiber from these consists mainly of unlignified cell walls. A representative of this wall type, prepared from immature cabbagE leaves, showed little ability to adsorb DNP. Two other cell-wall preparations, representing lignified walls of dicotyledons and unlignified walls of vegetative parts of grasses and cereals (monocotyledons belonging to the family Poaceae), adsorbed DNP much more effectively. However, two further preparations, representing suberized walls of cork cells and lignified walls of vegetative parts of grasses and cereals, were the most effective in adsorbing DNP. Extrapolation of these data to the in vivo situation would indicate that increased consumption of the vegetative parts of grasses or cereals and plant material containing cork cells, for example potato skins, could be effective in removing hydrophobic mutagens from potential contact with colonic mucosal cells.     

Functional differences between mammalian nuclear protein matrices and pore-lamina complex laminae

Nuclear matrix isolated from rat liver in the presence of a serine protease inhibitor contained 85% of the rapidly-labelled hnRNA, but pore-lamina preparations from the same tissue contained virtually none of the rapidly-labelled material. Nuclear matrix from rat endometrium, but not from lung, contained high-affinity binding sites for 17β-estradiol, but very few such sites were detected in endometrium pore-lamina preparations. It is concluded that the peripheral (porelamina) and intranuclear parts of the matrix differ functionally.     

A Rapid Method for Making Permanent Mounts of Nematodes

A rapid method which can be used to mount and clear nematodes and their eggs is presented. Permanent mounts of certain nematodes and parasite eggs have been prepared using a medium consisting of 56 parts of a stock solution of polyvinyl alcohol (“PVA”), 22 parts phenol and 22 parts of lactic acid. The stock solution of PVA is prepared by dissolving 15 grams of PVA in 100 ml. of distilled water. This medium can be used on material killed and fixed in 10% formalin, any concentration of alcohol, alcohol-glycerin or glycerin. Results have been very satisfactory in most instances. An accompanying plate of photographs shows some of the preparations obtained by using this method.     

Isotachophoretic and HPLC determination of nucleotides in rat liver cell nuclei isolated by non-aqueous technique

Rat liver whole cells and cell nuclei were prepared by a non-aqueous technique (glycerol). The nuclear preparations were of different purity as determined by RNA/DNA ratios (0.17-1.60) and accordingly were divided into 3 subgroups (mean values 0.29, 1.04 and 1.48). RNA nucleotides were separated by isotachophoresis and HPLC and calculated per mg DNA. Two of the nuclear subgroups (RNA/DNA = 1.04 and 1.48) had significantly elevated nucleotide values in relation to RNA/DNA. UDP-N-acetylhexosamine/DNA, on the contrary, was reduced in conformity with RNA in the preparations. Our findings may indicate different nucleotide concentrations in different parts of the cell.     

Essential light chain exchange in scallop myosin

The exchange of essential light chains (SH-LCs) of scallop myosin was followed with the aid of scallop SH-LC alkylated with 14C-labeled iodoacetate. More than 70% of the SH-LCs were exchanged in myosin preparations that were desensitized by removal of both regulatory light chains (R-LCs) with ethylenediaminetetraacetic acid (EDTA) treatment. Although desensitized myosin solubilized with 0.6 M NaCl or with 10 mM adenosine 5'-triphosphate (ATP) in the absence of salt equilibrated rapidly with SH-LCs even in the cold, exchange in myosin filaments required elevated temperatures. Equilibration of the SH-LCs in desensitized preparations did not depend on ATP or magnesium ions but was significantly accelerated by actin. The desensitized myosin preparations containing alkylated SH-LCs (approximately 1 mol of thiol alkylated/mol of SH-LC) readily recombined with R-LCs. The preparations regained fully the calcium dependence of the actin-activated magnesium adenosinetriphosphatase (Mg-ATPase), contained R-LCs and SH-LCs in equimolar amounts, and had an ATPase activity similar to that of untreated myosin preparations. R-LCs interfered with the equilibration of the SH-LCs. In intact myosin preparations, the exchange of SH-LCs was slow and was frequently associated with the dissociation of the R-LCs. The blocking action of the R-LC on SH-LC exchange agrees with evidence showing that the two light chain types interact and suggests that parts of the SH-LC may lie between the R-LC and the heavy chain of myosin.     

A New and Rapid Method for Making Permanent Aceto-Carmin Smears

Smear the pollen mother cells of a single anther from each flower bud on a clean dry slide, using a small scalpel. Flood the slide with Belling's acetocarmin and heat for a second over an alcohol flame. Examine under the microscope to determine the stage of microsporogenesis. If the stage is satisfactory, smear the remaining anthers in the same manner, but fix and stain them by immediate immersion, face downward, in a petri dish full of hot (steaming) acetocarmin for from 1 to 10 minutes. Then rapidly transfer thru the following mixtures: two parts 99% (glacial) acetic acid plus one part absolute ethyl alcohol; one part acetic acid plus two parts absolute alcohol; and finally one part acetic acid plus nine parts absolute alcohol. The slides are then to be dehydrated completely by 1 to 2 minutes immersion in pure absolute alcohol, and cleared 2 to 3 minutes in a mixture of xylene and absolute alcohol in equal parts. The preparations are then made permanent by mounting each with balsam and a cover glass. The whole process takes from 5 to 15 minutes and is particularly recommended for chromosome counts.     

Chemical composition and vasodilatation induced by Cuphea carthagenensis preparations

The aerial parts of Cuphea carthagenensis (Jacq.) J.F. Macbride (Lythraceae) are traditionally employed in Brazil to treat cardiovascular diseases. The aim of this study was to compare preparations of C. carthagenensis aerial parts (aqueous and ethanol extracts, together with derived fractions) with regard to their total phenolic contents and in vitro vasodilating activity. The main flavonoids found in the extracts were isolated and identified as quercetin derivatives. The extracts and fractions showed similar HPLC profiles with the presence of quercetin-5-O-β-glucopyranoside, quercetin-3-O-α-arabinofuranoside and quercetin-3-sulfate in all of them, but marked differences in the contents of flavonoids, proanthocyanidins, tannis and total phenolics. Excepting the aqueous extract, all assayed preparations elicited vasodilatation on pre-contracted rat aortic rings in the range of pIC(50) 4.53±0.03 to 4.98±0.06. Polynomial regression analysis demonstrated the relationship between vasodilating activity and the contents of flavonoids (r(2)=0.5190), proanthocyanidins (r(2)=0.8016), tannins (r(2)=0.8041) and total phenolics (r(2)=0.6226), suggesting the participation of these compounds in the pharmacological effect and their potential use as chemical markers for the species.     

Intrinsic organization of snake lateral cortex

Lateral cortex is the most laterally placed of the four cortical areas in snakes. Earlier studies suggest that it is composed of several subdivisions but provide no information on their organization. This paper first investigates the structure of lateral cortex in boa constrictors (Constrictor constrictor), garter snakes (Thamnophis sirtalis), and banded water snakes (Natrix sipedon) using Nissl and Golgi preparations; and secondly examines the relation of main olfactory bulb projections to the subdivisions of lateral cortex using Fink-Heimer and electron microscopic preparations. Lateral cortex is divided on cytoarchitectonic grounds into two major parts called rostral and caudal lateral cortex. Each part is further divided into dorsal and ventral subdivisions so that lateral cortex has a total of four subdivisions: dorsal rostral lateral cortex (drL), ventral rostral lateral cortex (vrL), dorsal caudal lateral cortex (dcL) and ventral caudal lateral cortex (vcL). Systematic analyses of Golgi preparations indicate that the rostral and caudal parts each contain distinct populations of neurons. Rostral lateral cortex contains bowl cells whose dendrites arborize widely in the outer cortical layer (layer 1). The axons of some bowl cells can be traced medially into dorsal cortex, dorsomedial cortex and medial cortex. Caudal lateral cortex contains pyramidal cells whose somata occur in layers 2 and 3 and whose dendrites extend radially up to the pial surface. In addition, three populations of neurons occur in both rostral and caudal lateral cortex. Stellate cells occur in all three layers and have dendrites which arborize in all directions. Double pyramidal cells occur primarily in layer 2 and have dendrites which form two conical fields whose long axes are oriented radially. Horizontal cells occur in layer 3 and have dendrites oriented concentric with the ependyma. Fink-Heimer preparations of snakes which underwent lesions of the main olfactory bulb show that the primary olfactory projections to cortex are bilateral and restricted precisely to rostral lateral cortex. Electron microscopic degeneration experiments indicate that the olfactory bulb fibers end as terminals which have clear, spherical vesicles and asymmetric active zones. The majority are presynaptic to dendritic spines in outer layer 1. These studies establish that lateral cortex in snakes is heterogeneous and contains two major parts, each containing two subdivisions. The rostral and caudal parts have characteristic neuronal populations. Primary olfactory input is restricted to rostral lateral cortex and seems to terminate heavily on the distal dendrites of bowl cells. Axons of some of these cells leave lateral cortex, so that the rostral lateral cortex forms a direct route by which olfactory information reaches other cortical areas. The functional role of caudal lateral cortex is not clear.     

Outer membrane proteins as major antigens of Fusobacterium nucleatum

The immunochemical reactions of rabbit polyclonal antibodies directed to different preparations of Fusobacterium nucleatum i.e, whole cells, peptidoglycan associated proteins, a peptidoglycan-protein complex and a purified 40 kiloDalton (kDa) protein, were investigated on outer membrane preparations of Fusobacterium species and a restricted number of Leptotrichia buccalis after their separation on sodium dodecyl sulphate polyacrylamide gels and electrotransfer to nitrocellulose. All F. nucleatum strains had identical reaction patterns with the immune sera tested. Surface exposed parts of a restricted number of proteins with apparent molecular weights at 70 kDa (a doublet band), 60 kDa, 55 kDa and 40 kDa seemed to be major immunogens. Antigenic related proteins either of identical or slightly deviating electrophoretic mobilities to the 40-kDa protein were observed with the other members of Bacteroidaceae tested. The characteristic 70-kDa protein doublet seemed to be restricted to F. nucleatum although single protein bands of near identical molecular weights belonging to the other species tested also reacted. The data also indicate that the 60-kDa and 55-kDa polypeptides might be present in other species of Fusobacterium.     

Outer membrane proteins as major antigens of Fusobacterium nucleatum

Abstract The immunochemical reactions of rabbit polyclonal antibodies directed to different preparations of Fusobacterium nucleatum i.e, whole cells, peptidoglycan associated proteins, a peptidoglycan-protein complex and a purified 40 kiloDalton (kDa) protein, were investigated on outer membrane preparations of Fusobacterium species and a restricted number of Leptotrichia buccalis after their separation on sodium dodecyl sulphate polyacrylamide gels and electrotransfer to nitrocellulose. All F. nucleatum strains had identical reaction patterns with the immune sera tested. Surface exposed parts of a restricted number of proteins with apparent molecular weights at 70 kDa (a doublet band), 60 kDa, 55 kDa and 40 kDa seemed to be major immunogens. Antigenic related proteins either of identical or slightly deviating electrophoretic mobilities to the 40-kDa protein were observed with the other members of Bacteroidaceae tested. The characteristic 70-kDa protein doublet seemed to be restricted to F. nucleatum although single protein bands of near identical molecular weights belonging to the other species tested also reacted. The data also indicate that the 60-kDa and 55-kDa polypeptides might be present in other species of Fusobacterium .     

Delafield's Hematoxylin and Safranin for Staining Meristematic Tissues

The following technic is suggested for staining permanent preparations of meristematic tissues: Prepare and mount the sections by the usual paraffin method. From water, stain them 2-10 minutes in a solution made by adding 2-4 cc. of Delafield's hematoxylin to a Coplin jar full of tap water. As staining is progressive, the sections should be examined from time to time with a microscope. When the cell walls have become a deep purple, transfer the preparations, thru the usual series, to a mixture of xylol-absolute-alcohol in equal parts, and from this to a counterstain made by adding 4-6 cc. of a saturated solution of safranin in absolute alcohol to a Coplin jar full of xylol (75%) with absolute alcohol (25%). This stains the nuclei. Leave the sections in the counterstain at least 2 hours and then rinse them in xylol-absolute-alcohol (1:1) to remove excess safranin. Transfer them to pure xylol and then mount them in neutral balsam.     

Sources of the blood supply to the human bulbar conjunctiva

In 93 complex preparations of the human orbit, the sources for blood supply of the eyeball conjunctiva, types of orbital vessels, including size of their diameters, were studied. The open parts of the conjunctiva were stated to get arteries of greater diameters comparing to those supplying the subconjunctival parts. It was noted that some sources of blood supply take their origin from the external carotid artery. This fact should be taken into account when choosing the part for biomicroscopy of the eyeball conjunctiva vessels.     

Prevalence and development of two Sarcocystis spp. in the horse (author's transl)

The prevalence of Sarcocystis spp. in horses was investigated in a survey at the Munich abattoir during 1978/79. Muscle specimens (oesophagus, diaphragm, sublingual muscle, myocardium) were examined using tryptic digestion. Out of 200 horses 31 (15.5%) were found to be carriers of sarcocysts. No parasites were found in the myocardium. In three animals sarcocysts could be isolated and differentiated in fresh preparations. Cysts with 5 to 11 microns by less than 0.5 microns hairlike, unstable protrusions were classified as Sarcocystis equicanis, whereas those with 2.5 to 4.5 microns by 0.8 to 1.0 microns fingerlike, stabile protrusions were assigned to be S. fayeri. Histologically S. equicanis cysts were thin-walled and S. fayeri cysts were thick-walled and often striated. For both species the dog acts as final host. A mixture of sporocysts of both species measured: 12.0--14.4 (13.4 +/- 0.7) X 9.3--10.5 (9.8 +/- 0.4) microns. The prepatent period is 11 to 17 days. Two ponies experimentally infected with 100,000 sporocysts each did not show clinical signs. In fresh preparations and in histopathological examinations of biopsied (111th, 130th, 152th, and 165th day post-infection (p.i.) and postmortem material (167th and 189th day p.i.) different developmental stages of sarcocysts of both species were seen and the following pathological alterations observed: circumscribed non-purulent inflammation, moderate Zenker's degeneration of muscle fibres, and degenerated cysts, of which sometimes only parts of the cyst wall were left. In fresh preparations S. equicanis and S. Fayeri could be differentiated 111 days p.i. The observed disappearance of the sarcocysts is suggested to be a self-cleaning process.     

Enzymatic conversion of dihydroflavonols to flavan-3,4-diols using flower extracts of Dianthus caryophyllus L. (carnation)

Flavonoid analysis and supplementation experiments with dihydroflavonols and leucocyanidin on two cyanic, two acyanic and one white/red-variegated flowering strain of Dianthus caryophyllus (carnation) showed that in the acyanic strains recessive alleles (aa) of the gene A interrupt the anthocyanin pathway between dihydroflavonols and leucoanthocyanidins. The instability in the variegated strain involves the same step and is obviously caused by the multiple allele a ^var . In confirmation of these results, dihydroflavonol 4-reductase activity could be demonstrated in enzyme extracts from cyanic flowers and cyanic parts of variegated flowers but not in preparations from acyanic flowers or acyanic parts. The enzyme catalyzes the stereospecific reduction of (+)dihydrokaempferol to (+)-3,4-leucopelargonidin with NADPH as cofactor. A pH optimum around 7.0 and a temperature optimum at 30° C was determined, but the reduction reaction also proceeded at low temperatures. (+)Dihydroquercetin and (+)dihydromyricetin were also reduced to the respective flavan-3,4-cis-diols by the enzyme preparations from carnation flowers, and were even better substrates than dihydrokaempferol.These investigations were supported by grants from Fonds zur Förderung der wissenschaftlichen Forschung and Deutsche Forschungsgemeinschaft. The authors thank the market-gardens Ing. K. Rungaldier (Vienna, Austria), A. Sinner (Tübingen, FRG) and Barbaret & Blanc GMBH (Horhausen, FRG) for generous support with plant material.     

Isolation of a new germ line-specific repetitive DNA family in Acricotopus by microdissection of polytenized germ line-limited chromosome sections from a permanent larval salivary gland preparation

In Acricotopus lucidus (Diptera, Chironomidae) the germ line-limited chromosomes (Ks) have developed from the soma chromosomes (Ss) by endoreduplication, rearrangements and accumulation of germ line-specific repetitive sequences. For molecular analysis of specific small K sections, microdissection of metaphase Ks generally yields very limited amounts of DNA. In this study, K-specific DNA was microdissected from defined polytenized K sections of X-ray induced K-S-rearrangements of permanent salivary gland chromosome preparations and was then amplified by DOP-PCR. A new germ line-specific tandem repetitive DNA family was isolated by this way from a heterochromatic K segment, characterized and localized on the Ks by FISH. The repetitive elements are related to sequences of earlier described K-specific tandem repetitive DNA families in A. lucidus, but are located mainly in terminal heterochromatin bands of the two largest Ks and only to a limited degree in the paracentromeric K heterochromatin. This demonstrates that a collection of permanent preparations of K-S-rearrangements with polytenized heterochromatic and S-homologous K sections of A. lucidus can be used as a source for obtaining K sequences of defined K parts to investigate the molecular evolution of the Ks.     

Spectral analysis of chromatin and its components in the vacuum ultraviolet region of the spectrum

Electronic absorption spectra of thin films of chromatin and chromatin components in ultraviolet (140-280 nm) were investigated. The absorption coefficients mu (lambda) of chromatin, nucleosomes with and without histone H1, total histones (TH), DNA were compared. The spectra of nucleosomes and chromatin differ from summary spectra of DNA + TH. The lack of additivity of absorption coefficients at different wavelengths may be explained by different conformational changes of free DNA, TH and DNA, TH in nucleosomes and chromatin during the process of drying aqueous solutions for the preparations of thin films. The obtained mu (lambda) values are necessary for the estimation of the DNA and TH parts of absorption in chromatin and nucleosomes in the investigations of UV and VUV irradiation damages.     

Insect olfactory coding and memory at multiple timescales

Insects can learn, allowing them great flexibility for locating seasonal food sources and avoiding wily predators. Because insects are relatively simple and accessible to manipulation, they provide good experimental preparations for exploring mechanisms underlying sensory coding and memory. Here we review how the intertwining of memory with computation enables the coding, decoding, and storage of sensory experience at various stages of the insect olfactory system. Individual parts of this system are capable of multiplexing memories at different timescales, and conversely, memory on a given timescale can be distributed across different parts of the circuit. Our sampling of the olfactory system emphasizes the diversity of memories, and the importance of understanding these memories in the context of computations performed by different parts of a sensory system.