X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

Proliferation and germination of somatic embryos from embryogenic suspension cultures in <Emphasis Type="Italic">Phœnix dactylifera</Emphasis>

The present study demonstrates a procedure for the rapid development of a high number of somatic embryos from embryogenic suspension culture. This method might be efficient for mass propagation of Phnix dactylifera L. Embryogenic callus placed in liquid medium with 10^–5M ABA yielded an average 72 embryos per 100ml of culture medium within 2months, while those placed on solid medium yielded an average of 33, 20 and 16 embryos per 100ml of culture medium respectively for 10^–7, 10^–6 and 10^–5 M ABA after 4months. The combination of 2,4-DIchlorophenoxyacetic acid (2,4-D) (4.5×10^–7M), glutamine (6.7×10^–4M), and ABA (10^–5M) (L8 liquid medium) showed a beneficial effect on somatic embryos production compared to 2,4-D and glutamine alone, while this combination significantly (p<0.05) increased the accumulation of storage proteins (144 and 138mgg^–1 DW respectively for Jihel and Bousthami noir cultivars) in somatic embryos. The somatic embryos which underwent maturation on medium containing only 4.5×10^–7M 2,4-D and 10^–5M ABA (L6 liquid medium) accumulated more sugars (292 and 265mgg^–1 DW respectively for Jihel and Bousthami noir) than those matured on any other liquid medium. Histological studies revealed that somatic embryos (developed in L6 and L8 liquid media) accumulated less reserve compounds (proteins and sugars) than zygotic embryos. The addition of activated charcoal (0.25 and 0.5gl^–1) and phytagel^® (2.5gl^–1) to the germination medium may be useful for enhancing the germination of Phnix dactyliferasomatic embryos.     

Macronutrient input from pollen in two regenerating pine stands in southeast Korea

This study examined macronutrient input from pollen in two naturally regenerating pine stands in southeast Korea. Durham gravity pollen collectors were used to measure pine pollen deposition and the macronutrients in the collected pine pollen were analyzed. In 1998, pine pollen deposition began just before 18 April and lasted for approximately 2weeks. Total pine pollen deposition differed between the two sampling sites; 27.5kgha^–1 was collected from the mature stand and 17.7kgha^–1 was collected from the young stand. The values for nutrient deposition from pine pollen are 549gha^–1 N, 78gha^–1 P, 240gha^–1 K, 45gha^–1 S and 22gha^–1 Mg at the mature stand and 353gha^–1 N, 51gha^–1 P, 151gha^–1 K, 27gha^–1 S and 14gha^–1 Mg at the young stand, suggesting that nutrients from pine pollen contribute to forest nutrient cycling. The pine pollen deposition values obtained from our study (17.7–27.5kg^–1ha^–1year^–1) are approximately 1/115–180-fold that of pine litterfall in Korea. If we take pollen nutrients into account, the contribution rate of pollen to the annual nutrient input is very high in our study (N 1/30, P 1/5, K 1/9 that of litterfall). Macronutrient deposition from pine pollen is concentrated temporally in spring. Although the annual contribution of nutrient mass by pollen is small compared to that of litterfall, the rapid turnover rate of pollen nutrients combined with episodic deposition suggests that pollen may play a disproportionate role in temperate pine forest nutrient cycling.     

Characterization of Baboon (Papio hamadryas) Milk Proteins

The major proteins of baboon milk were identified as -lactoglobulin (LG), -lactalbumin (LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human LA, lysozyme, and albumin and bovine LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon LG are identical to those of macaque (Macaca fasicularis) LG except for a (D/N) polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of LG were elucidated using RT-PCR amplification of poly(A)⁺ mRNA purified from lactating mammary gland. Baboon LG consists of 168 amino acid residues (M_r 20,750) and is the longest LG identified to date. LG and LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4–6, of individual baboon milk samples at varying stages of lactation.     

A non-specific Ca2+ (or Mg2+)-stimulated ATPase in rat heart sarcoplasmic reticulum

ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Increasing concentrations of Mg²⁺ produced an apparent inhibition of the Ca²⁺-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca²⁺ with a high affinity (K_0.5 Ca²⁺ = 0.41 M) suggesting the presence of a high affinity Ca²⁺-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations and failed to stimulate membrane phosphorylation and Ca²⁺ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48180 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca²⁺-pumping ATPase previously characterized in dog heart and other species.Abbreviations cAMP adenosine 3,5-monophosphate - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - PLB phospholamban - SR sarcoplasmic reticulum - TFP trifluoperazine     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.     

Sparse distribution of γ/δ T lymphocytes around human epithelial tumors predominantly infiltrated by primed/memory T cells

Summary Evidence from the mouse system has suggested that T lymphocytes accumulating in non-lymphoid tissue, in particular epithelia, may preferentially express the T cell receptor (TCR) . In this study, we characterize the T cell receptor or phenotype of lymphocytes infiltrating human tumors of epithelial origin using monoclonal antibodies (mAb) for immunohistology and flow cytometry on cells extracted by enzyme digestion. This report shows that the majority of CD3⁺ tumor-infiltrating lymphocytes are TCR ⁺ but a small percentage of TCR can be clearly defined scattered throughout the tumor tissue with apparently no microanatomical selection. So far there has been little evidence for an accumulation of activated T cells in human tumor tissues as defined by mAb against molecules appearing transiently during the acute phase of activation. Now mAb are available that can identify primed or memory T cells such as mAb UCHL-1 recognizing the CD45RO antigen. Here we show that CD3⁺ tumor-infiltrating lymphocytes have a statistically significant accumulation of primed T cells, as compared to the autologous peripheral blood lymphocytes, suggesting their immune stimulation by tumor cells.     

Analysis of primary food sources and trophic relationships of aquatic animals in a mangrove-fringed estuary,Khung Krabaen Bay (Thailand) using dual stable isotope techniques

Stable carbon (¹³C) and nitrogen (¹⁵N) isotopes were used to elucidate primary food sources and trophic relationships of organisms in Khung Krabaen Bay and adjacent offshore waters. The three separate sampling sites were mangroves, inner bay and offshore. The ¹³C values of mangrove leaves were –28.2 to –29.4, seagrass –10.5, macroalgae –14.9 to –18.2, plankton –20.0 to –21.8, benthic detritus –15.1 to –26.3, invertebrates –16.5 to –26.0, and fishes –13.4 to –26.3. The ¹⁵N values of mangrove leaves were 4.3 to 5.7, seagrass 4.3, macroalgae 2.2 to 4.4, plankton 5.7 to 6.4 , benthic detritus 5.1 to 5.3, invertebrates 7.2 to 12.2 , and fishes 6.3 to 15.9. The primary producers had distinct ¹³C values. The ¹³C values of animals collected from mangroves were more negative than those of animals collected far from shore. The primary carbon sources that support food webs clearly depended on location. The contribution of mangroves to food webs was confined only to mangroves, but a mixture of macroalgae and plankton was a major carbon source for organisms in the inner bay area. Offshore organisms clearly derived their carbon through the planktonic food web. The ¹⁵N values of consumers were enriched by 3–4 relative to their diets. The ¹⁵N data suggests that some of aquatic animals had capacity to change their feeding habits according to places and availability of foods and as a result, individuals of the same species could be assigned to different trophic levels at different places.     

Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoproteins

Structures of acidic N-glycans released from porcine zona pellucida glycoproteins by hydrazinolysis were studied. The results indicated that the acidic glycans are of mono- to tetraantennary complex-type with and without N-acetyllactosamine repeating units. Sulfated residues are not only located at the C-6 position of GlcNAc included in the N-acetyllactosamine repeating units, but also at the C-6 position of GlcNAc in the non-repeated antennae and at the C-3 position of reducing terminal GlcNAc residue. Analysis of the oligosaccharide fragments released by endo--galactosidase digestion and by hydrazine/nitrous acid treatment also revealed that various sulfated and non-sulfated forms of fucosylated structures such as Fuc12Gal14(±SO–36)GlcNAc (type 2H), Gal14(Fuc13)(±SO–36)GlcNAc(Lex) and Fuc13 or 4(±SO–36)GlcNAc, are expressed in the repeated outer chain moieties.     

Quantification of soil carbon inputs under elevated CO2: C3 plants in a C4 soil

The objective of this investigation was to quantify the differences in soil carbon stores after exposure of birch seedlings (Betula pendula Roth.) over one growing season to ambient and elevated carbon dioxide concentrations. One-year-old seedling of birch were transplanted to pots containing C₄ soil derived from beneath a maize crop, and placed in ambient (350 L L^–1) and elevated (600 L L^–1) plots in a free-air carbon dioxide enrichment (FACE) experiment. After 186 days the plants and soils were destructively sampled, and analysed for differences in root and stem biomass, total plant tissue and soil C contents and ¹³C values. The trees showed a significant increase (+50%) in root biomass, but stem and leaf biomasses were not significantly affected by treatment. C isotope analyses of leaves and fine roots showed that the isotopic signal from the ambient and elevated CO₂ supply was sufficiently distinct from that of the C₄ soil to enable quantification of net root C input to the soil under both ambient and elevated CO₂. After 186 days, the pots under ambient conditions contained 3.5 g of C as intact root material, and had gained an additional 0.6 g C added to the soil through root exudation/turnover; comparable figures for the pots under elevated CO₂ were 5.9 g C and 1.5 g C, respectively. These data confirm the importance of soils as an enhanced sink for C under elevated atmospheric CO₂ concentrations. We propose the use of C₄ soils in elevated CO₂ experiments as an important technique for the quantification of root net C inputs under both ambient and elevated CO₂ treatments.     

Sequential conversion of cortexolone to prednisolone by immobilized mycelia of Curvularia lunata and immobilized cells of Arthrobacter simplex

Summary Two-step bioconversion of cortexolone (Reichstein's Compound S) to its ¹-dehaydro-11-hydroxy derivative, prednisolone, was successfully performed by the combined use of immobilized Curvularia lunata mycelia and immobilized Arthrobacter simplex cells. Immobilized living mycelia of C. lunata having a high 11-hydroxylation activity were prepared by in situ germination of spores entrapped in photo-crosslinked resin gels of a suitable net-work structure. Acetone-dried cells of A. simplex having an induced steroid ¹-dehydrogenase activity were also entrapped with photo-crosslinkable resin prepolymers and used for - dehydrogenation of hydrocortisone to prednisolone. For the production of prednisolone from cortexolone, the combination of sequential steps, 11-hydroxylation and subsequent ¹-dehydrogenation, was found to be suitable. Each immobilized microbial cell system was stable and could be used for the sequential reactions repeatedly (operational period, 25 days).     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

Simplified typing of mouse hemoglobin (Hbb) phenotypes using cystamine

Cellulose acetate electrophoresis of mouse hemoglobins modified with the disulfide reagent cystamine permits rapid, unequivocal discrimination of all combinations of the codominant mouse hemoglobin single (Hbb ^s ) and diffuse (Hbb ^d and Hbb ^p ) alleles. The single, diffuse major, diffuse d-minor, and diffuse p-minor adult hemoglobins are all resolved by this method, which depends on the presence of a cysteine in the chains of diffuse mice which is not found in the chain of single mice.This work was supported by research grants ACS-VC58 and NIH CA-01074. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Uneven Distribution and Size of Rabbit Lens Capsule Proteoglycans

To document the ultrastructural distribution of lens capsule proteoglycans, rabbit lens capsules were fixed and stained overnight in 50mM sodium acetate, pH 5.6, containing 2.5% glutaraldehyde, 0.2% Cuprolinic Blue and 0.2M MgCl2. They were rinsed, stained with 1% aqueous sodium tungstate, embedded in Epon, sectioned (60nm), and examined with an electron microscope at 60kV.Proteoglycan–Cuprolinic Blue complexes mainly appeared as networks of small electron-dense filaments throughout the posterior and anterior capsules. The posterior capsule was a single layer with a network of small proteoglycan filaments gradually decreasing in size from the humoral side (90×10nm) to the lenticular side (30×8nm). The humoral side of the anterior capsule had a thin lamina (400nm) containing large (180×40nm), very electron-dense proteoglycan–Cuprolinic Blue complexes plus small proteoglycans. Below this lamina, the complexes were only seen as filaments slightly smaller than those in the corresponding area of the posterior capsule.Cuprolinic Blue binding of the anterior and posterior lens capsules revealed differences in the size and distribution of their sulphated proteoglycans which do not correspond to the patterns of their immunoreactivity with anti-heparan sulphate proteoglycan. The humoral lamina in the anterior capsules, with large proteoglycan structures, might be a distinct structural and functional compartment.     

Microphotometric determination of enzyme activities in type-grouped fibres of reinnervated rat muscle

Summary Activities of succinate dehydrogenase (SDH), glycerolphosphate oxidase (GP-OX), cytochrome oxidase (CYT-OX) and lactate dehydrogenase (LDH) were determined microphotometrically in single, actomyosin-ATPase typed (Guth and Samaha 1970) fibres within cross-sections of normal and reinnervated rat tibialis anterior muscles. SDH and GP-OX activities displayed pronounced scattering and large overlaps existed between -, -, and -fibres of normal muscle. Coefficients of variation were in the range of 16–40% for GP-OX and SDH in the different fibre populations. Enzyme activity determinations in typegrouped -, -, and -fibres of reinnervated muscle showed much less scattering than in normally distributed -, -, and -fibres of control muscles. Coefficients of variation were in the range of 10–13% for SDH, GP-OX, CYT-OX and LDH. The experimental error of the kinetic microphotometric measurement of enzyme activities in situ is in the range of 10% (Reichmann and Pette 1982). Our results therefore suggest a high degree of metabolic similarity or homogeneity of typed-grouped muscle fibres and thus support the assumption that type-groped fibres are homogeneous and correspond to regularly assembled motor units.     

Soret spectroscopic and molecular graphic analysis of human semi-beta-hemoglobin formation

The interaction of heme-free (^o) and heme-containing (^h) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10⁷ M^–1 s^–1. This was 100-fold more rapid than the reported rate of combination of ^h and ^h proteins. This encounter-like rate of semi--hemoglobin (^oh) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled ^o protein superimposed over native ^h protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the ₁₁ interface, was apparently responsible for the enhanced association properties of the ^o protein.     

Vertical planktonic structure in the central basin of Lake Baikal in summer 1999, with special reference to the microbial food web

Planktonic microbial interactions in the central basin of Lake Baikal were examined on a summer day in 1999. The subsurface maxima of bacterial abundance and chlorophyll concentration were recorded at the same depth, whereas the vertical distribution of heterotrophic nanoflagellates was the inverse of those of bacteria and picophytoplankton. Release of extracellular organic car-bon (EOC) from phytoplankton was estimated by the NaH¹⁴CO₃ method as 2.4µgCl^–1day^–1. Bacterial production (4.3µgCl^–1day^–1), estimated in a bottle incubation experiment using size-fractionated water samples, exceeded the EOC released. Thus, other supplying sources of organic matter are needed for the bacterial production. Grazing (2.6µgCl^–1day^–1) was also estimated in the experiment and accounted for 60% of the bacterial production. This is the first report on the microbial food web in the central basin of Lake Baikal.     

Fine root biomass,production and turnover in a fertilized <Emphasis Type="Italic">Larix leptolepis</Emphasis> plantation in central Korea

The effects of fertilization [control (C), 200kgNha^–1+25kgP ha^–1 (LNP) and 400kgNha^–1+ 50kgP ha^–1 (HNP)] on fine root dynamics were examined in a 40-year-old Larix leptolepis plantation in central Korea. The average fine root biomass during the growing season for C, LNP and HNP was 957, 934 and 814kgha^–1, respectively, whereas the fine root production for C, LNP and HNP was 2103, 2131 and 2066kgha^–1, respectively. Nitrogen and P inputs into the soil via fine root turnover for C, LNP and HNP were 23.0 and 1.2, 23.3 and 1.2 and 22.6 and 1.2kgha^–1, respectively. There were no significant differences in fine root biomass, production and N and P inputs through fine root turnover between the fertilization treatments during the first growing season after fertilization.     

The solution structure of rat Aβ-(1–28) and its interaction with zinc ion: insights into the scarcity of amyloid deposition in aged rat brain

The amyloid -peptide (A) is a major component of insoluble amyloid deposits in Alzheimers disease, and the ability of the -peptide to exist in different conformations is dependent on residues 1–28 [-(1–28)]. However, different from humans, no A amyloid deposition has been found in aged rats brains. Studying the three-dimensional solution structure of rat A-(1–28) and the binding circumstance of Zn²⁺ is beneficial to a clear understanding of the potential role of Zn²⁺ in Alzheimer-associated neuropathogenesis and to suggest why there is no amyloid deposition in aged rats brains. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of rat A-(1–28) and the binding constant of Zn²⁺ to rat A-(1–28). Our results suggest that (1) the three-dimensional solution structure of rat A-(1–28) is more stable than that of human A-(1–28) in DMSO-d₆ and that a helical region from Glu16 to Val24 exists in the rat A-(1–28); (2) the affinity of Zn²⁺ for rat A-(1–28) is lower than that for human A-(1–28) and the NMR data suggest that Arg13, His6, and His14 residues provide the primary binding sites for Zn²⁺; and (3) the proper binding of Zn²⁺ to rat A-(1–28) can induce the peptide to change to a more stable conformation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0556-xAbbreviations A amyloid -peptide - AD Alzheimers disease - hA-(1–28) human A-(1–28) - rA-(1-28) rat A-(1–28) - REM restrained energy minimization