Electron microscopy of supercoiled pEJ4 DNA containing homopurine.homopyrimidine sequences

Supercoiled pEJ4 DNA (a derivative of pUC19 containing an insert with 60-bp-long homopurine.homopyrimidine tract from the sea urchin P. miliaris histone gene spacer) was investigated by electron microscopy using three different spreading techniques i.e., formamide and aqueous variants of the Kleinschmidt technique and protein-free benzyldimethyl-alkyl ammonium chloride (BAC) technique at different pHs. If the specimens for electron microscopy were prepared at pH 5.6 and pH 4.0 (i.e., under conditions where the homopurine.homopyrimidine tract assumes an unusual conformation) a single thick "stem" or a "denaturation bubble" in a large number of DNA molecules were observed. No such changes were found in samples prepared at neutral pH and in linearized pEJ4 DNA prepared at pH 5.6. In specimens of a control supercoiled pUC19 DNA prepared at pH 5.6 and 4.0 practically no local changes were detected. The "denaturation bubbles" were observed by BAC techniques (probably due to secondary local DNA denaturation during the specimen preparation) while the more gentle formamide technique revealed only "stems". The "stems" were almost always positioned at the sites where the curvature of supercoiled DNA molecules occurred. The results are in agreement with presence of a protonated triplex H-form in homopurine.homopyrimidine tract bringing the first evidence of curvature or kinking of the DNA molecule connected with the occurrence of the H-form in supercoiled DNA.     

Stand microclimate and physiological activity of tree leaves in an oak-hornbeam forest

Summary In an uneven-aged, multi-species oak-hornbeam forest at Báb, SW Slovakia (former IBP Forest Research Site), a series of micrometeorological and ecophysiological measurements started in 1985. The aims of the work are to improve understanding of physiological processes (photosynthesis, respiration, and transpiration) of adult trees and stand microclimate, to collect data for simulation of the canopy (stand) photosynthesis and for ecological synthesis of the functioning of the forest ecosystem. In this paper, photosynthetically active radiation (PAR), air temperature (AT) and relative humidity (RH), wind speed (WS), and CO₂ concentration ([CO₂]) in and above the forest are characterized for the fully leaved season, using diurnal courses, vertical profiles and isodiagrams (isopleths). Approximately 50% of incident PAR was absorbed by the upper 4–5 m layer of leaves and only approximately 5% or less penetrated to the forest floor. Vertical gradients of AT and RH were generally low, but large differences in diurnal ranges of AT and RH were observed between vertical levels. The upper leaf canopy greatly reduced WS, and at a height of about 14 m above the ground it was close to zero. The highest diurnal [CO₂] maximum and variations occurred at 1 m above the ground, and the lowest above the forest. In good light conditions in the forest, the entire leaf canopy (overstorey and understorey canopy) is a large sink of CO₂. At night the forest stand is a source of CO₂, the largest internal source being the soil and forest floor.     

Histochemical demonstration of purine nucleoside phosphorylase (PNPase) in microglial and astroglial cells of adult rat brain

The histochemical localization of enzymes associated with purine nucleoside metabolism indicates that glial cells might participate in the regulation of these compounds in the central nervous system. In the present study we examined the histochemical localization of purine nucleoside phosphorylase (PNPase) in sections from adult rat brain. Some sections were also sequentially stained immunocytochemically for astroglial or microglial cells utilizing glial fibrillary acidic protein (GFAP) or OX-42 antibodies, respectively. Our observations showed that PNPase was restricted to glial cells, whereas neurons always remained negative. Brain sections stained for both PNPase and GFAP showed that the GFAP-positive astroglial cells were always PNPase positive. Other PNPase-positive but GFAP-negative cells were also observed. These cells resembled microglial cells, and brain sections reacted for both PNPase and OX-42 confirmed this by showing that the major part of OX-42-positive microglial cells were PNPase positive. In these sections, the PNPase-positive but OX-42-negative cells present resembled astroglial cells. From our double staining experiments, we conclude that PNPase is present in both astroglial and microglial cells in normal adult brain.     

A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-gamma/delta+ T lymphocytes. I. Inhibition of the IL-2-dependent proliferation by anti-Kp43 monoclonal antibody.

In the present study we describe a novel functional cell surface molecule, designated as Kp43, which is expressed among leukocytes by NK cells, TCR-gamma/delta + T lymphocytes, and some CD8+ CD56+TCR-alpha/beta + T cell clones. The Kp43 Ag is a 70-kDa disulfide-linked dimer, which migrates in SDS-PAGE under reducing conditions as a single 43-kDa band. Two-color immunofluorescence staining of fresh PBL revealed that only a fraction of CD16+, and of TCR-gamma/delta + T lymphocytes expressed the Ag. The analysis of TCR-alpha/beta + T cell clones showed that a small proportion (2 out of 20) weakly expressed Kp43 together with the CD8 and CD56 molecules. By immunoperoxidase staining of different tissues the anti-Kp43, reactivity was detected exclusively in lymphoid organs, where a minority of scattered cells was stained, and in some liver sinusoidal cells. Essentially all NK cells acquired Kp43 when stimulated with a B lymphoblastoid cell line. By contrast, the pattern of distribution of Kp43 remained stable upon in vitro culture of T-gamma/delta lymphocytes, thus delineating two subsets according to its expression. In lymphokine-activated killer populations, obtained by culturing either PBL or NK cells with high concentration of IL-2, most CD16+ and CD56+ cells became Kp43+. The Kp43-specific mAb inhibited the IL-2-dependent proliferative response of cultured NK and TCR-gamma/delta + T cells without affecting their non-MHC-restricted cytotoxicity. The partial inhibitory effect, which was mediated as well by pepsin digested F(ab')2 fragments, was lost upon reduction to Fab. The anti-Kp43 mAb did not interfere with the specific binding of IL-2 to its surface receptors. Altogether the data point out that the Kp43 dimer is involved in the regulation of the IL-2-dependent proliferative response of NK cells and a subset of TCR-gamma/delta + T lymphocytes.     

Mixed infection of Rickettsiella phytoseiuli and Coxiella burnetii in Dermacentor reticulatus female ticks: electron microscope study

Mixed infection of Rickettsiella phytoseiuli and Coxiella burnetii was investigated in hemolymph and organs of experimentally infected females of Dermacentor reticulatus ticks. Following intracoelomic infection, both agents, with the exception of Gene's organ, multiplied well in the cells of the tick host's organs. Two out of six developmental stages of R. phytoseiuli, i.e., crystal-forming and small dark particles, in dual infection with C. burnetii revealed marked morphological alterations. C. burnetii in the presence of R. phytoseiuli penetrated into the cortical layer of the synganglion and into the alveoli of the second and third type of salivary glands, but did not occur in the single infection.     

Histidine 21 is at the NAD+ binding site of diphtheria toxin

Treatment of fragment A chain of diphtheria toxin (DT-A) with diethylpyrocarbonate modifies His-21, the single histidine residue present in the chain, without alteration of other residues. Parallel to histidine modification, NAD+ binding and the NAD-glycohydrolase and ADP-ribosyltransferase activities of DT-A are lost. Both NAD+ and adenosine are very effective in protecting DT-A from histidine modification and in preserving its biological properties, while adenine is ineffective. Reversal of histidine modification with hydroxylamine restores both NAD+ binding and enzymatic activities of the toxin. The possible role of His-21 in the activity of diphtheria toxin is discussed in relation to the available three-dimensional structure of the related toxin produced by Pseudomonas aeruginosa.     

Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43

Growth cones, the motile apparatus at the ends of elongating axons, are sites of extensive and dynamic membrane-cytoskeletal interaction and insertion of new membrane into the growing axon. One of the most abundant proteins in growth cone membranes is a protein designated GAP-43, whose synthesis increases dramatically in most neurons during periods of axon development or regeneration. We have begun to explore the role of GAP-43 in growth cone membrane functions by asking how the protein interacts with those membranes. Membrane-washing experiments indicate that mature GAP-43 is tightly bound to growth cone membranes, and partitioning of Triton X-114-solubilized GAP-43 between detergent-enriched and detergent-depleted phases indicates considerable hydrophobicity. The hydrophobic behavior of the protein is modulated by divalent cations, particularly zinc and calcium. In vivo labeling of GAP-43 in neonatal rat brain with [35S]methionine shows that GAP-43 is initially synthesized as a soluble protein that becomes attached to membranes posttranslationally. In tissue culture, both rat cerebral cortex cells and neuron-like PC12 cells actively incorporate [3H]palmitic acid into GAP-43. Isolated growth cones detached from their cell bodies also incorporate labeled fatty acid into GAP-43, suggesting active turnover of the fatty acid moieties on the mature protein. Hydrolysis of ester-like bonds with neutral hydroxylamine removes the bound fatty acid and exposes new thiol groups on GAP-43, suggesting that fatty acid is attached to the protein's only two cysteine residues, located in a short hydrophobic domain at the amino terminus. Modulation of the protein's hydrophobic behavior by divalent cations suggests that other domains, containing large numbers of negatively charged residues, might also contribute to GAP-43-membrane interactions. Our observations suggest a dynamic and reversible interaction of GAP-43 with growth cone membranes.     

Allozyme variation and differentiation in African and Indian rhinoceroses

We studied variation at 25 to 31 allozymic loci in African and Asian rhinoceroses. Four taxa in three genera were examined: African Ceratotherium simum simum (northern white rhinoceros), C. s. cottoni (southern white rhinoceros), Diceros bicornis (black rhinoceros), and Rhinoceros unicornis (Indian rhinoceros). Extremely small amounts of intraspecific variation were observed in sample sizes of 2 to 10 presumably unrelated individuals per taxon: P = .00-.10, H = 0.00-0.02. We examined demographic bottlenecks and sampling errors as possible reasons for the low levels of detectable variation. The very small intraspecific genetic distance (D = 0.005) between the two living white rhinoceros subspecies is far less than the distance that has been reported for other mammal subspecies. The mean D value of 0.32 +/- 0.11 between the two African genera was also less than expected given the divergence time of greater than 7 million years suggested by the fossil record. Rhinoceroses may be evolving more slowly at the structural gene loci than are some other mammal groups. The estimate of D = 1.05 +/- 0.24 for the African-Indian split supports this idea, as the lineage diverged at least 26 million years ago. Our results contribute to the currently available scientific information on which management decisions aimed toward saving endangered rhinoceroses should be based.