Studies of the morphology of chromosomes of some selected species

Karyotypes of twelve species from twenty-four localities in southern Moravia and one locality in Slovakia were investigated. Their counts or karyotypic formulae are as follows:Chenopodium foliosum (Moench) Ascherson: K (2n)=18=16 A^m+2 B^sm;Astragalus austriacus Jacq.: K (2n)=16=8 A^m+8 B^sm;Astragalus exscapus L.: K (2n)=16=10 A^m+4 B^sm+2 C^st;Astragalus cicer L.: K (2n)=64;Astragalus onobrychis L.: K (2n=64 and K (2n)=64+1;Vicia dumetorum L.: K (2n=14=10 A^m+4 B^sm;Vicia sylvatica L.: K (2n)=14=2 A^m+10 B^sm+2 C^st;Vicia pisiformis L.: K (2n)=12=8 A^m+4 B^sm;Vicia cassubica L.: K (2n)=12=4 A^m+6 B^sm+2 C^st;Vicia cracca L. (from five localities in southern Moravia): K (2n)=28=4 A^m+12 B^sm+12 C^st and K (2n)=28+1=5 A^m+12 B^sm+12 C^st;Vicia cracca L. (from one locality in Slovakia): K (2n)=14=2 A^m+6 B^sm+6 C^st;Vicia tenuifolia Roth: K (2n)=24=4 A^m+16 B^sm+4 C^st;Serratula lycopifolia (Vill.) Kern.: K (2n)=60.     

Molecular cytogenetic studies of the “Xanthocephalum group” (Asteraceae)

Fourteen North American members of the “Xanthocephalum group” were studied by classical and molecular cytogenetics. Location and number of rDNA sites were determined by FISH. For the 5S rDNA, a probe was obtained from Prionopsis ciliata. Most species were diploid (2n?=?12), although Isocoma menziesii, Grindelia hirsutula, G. robusta, both varieties of G. stricta, and one population of G. camporum were tetraploid (2n?=?24). Diploid Grindelia and Prionopsis ciliata were 5m?+?1sm, tetraploids 10m?+?2sm, except G. hirsutula (8m?+?4sm), and Isocoma and Olivaea 6m?+?2sm and 3m?+?3sm, respectively. Most species had satellites on the short arms of m pairs: two in tetraploids and P. ciliata and one in diploids. Satellites were associated with two CMA⁺/DAPI^? bands in diploid species and four bands in tetraploids and in P. ciliata. rDNA loci (two in diploids to four in tetraploids) may be indicative of ploidy level. Grindelia tetraploids could have originated recently by autopolyploidy. Chromosome duplication was followed by modifications in the genome structure, resulting in higher heterochromatin amounts not associated with NORs. There is only one 5S site per basic genome in para or pericentromeric regions. Although not always large, chromosome variation has accompanied the evolutionary divergence of the taxa studied.     

Mapability of Very Close Markers of Bacteriophage λ

Recombinant frequency was compared with nucleotide distance in crosses involving markers in either the P_RM or the cy region of phage λ. For each pair of markers, we performed reciprocal four-factor crosses of the following types: (I) A⁺m₁ ⁺m₂^-B^- x A^-m₁ ^-m₂⁺B⁺; and (II) A⁺m₁ ^-m₂⁺B^- x A^-m₁ ⁺m₂^-B⁺. In crosses of type I, the frequency of A⁺m₁ ⁺m₂⁺B⁺ recombinants among total (selected) A⁺B⁺ progeny was directly proportional to nucleotide distance between m₁ and m₂ in the range from 3 to 160 nucleotides. When less than three nucleotides separated m₁ and m₂, the measured yields of m₁⁺m₂⁺ recombinants were significantly depressed.

We also found that the frequency of A⁺m₁ ⁺m₂⁺B⁺ recombinants among total A⁺B⁺ progeny was significantly lower (about 10-fold on the average) in crosses of type II than in the corresponding crosses of type I. Since mismatch correction should yield A⁺m₁ ⁺m₂⁺B⁺ recombinants with approximately equal frequencies in type I and II crosses, we suggest: (1) that most m₁⁺m₂⁺ recombinants produced in type I crosses must arise from the formation of heteroduplex structures with a discontinuity (in the source of genetic information) between sites m₁ and m₂, and (2) that mismatch correction is not a major pathway for production of recombinants for close markers in normal λ infection.

     

A contribution to the study ofChenopodium album agg

Plants gathered in Staré Brno (1979)—classified under the genusChenopodium L. and having K (2n)=54—are hybrids.Chenopodium strictum Roth (n=18) andChenopodium suecicum J. Murr (n=9) are probably parental species. Hybridization may have been linked with amphidiploidy.     

Karyology of theScorzonera (Compositae) species from the Iberian Peninsula

A karyological study of 15 taxa ofScorzonera L. from the Iberian Peninsula has been made. The chromosome numbers found inS. hispanica var.pinnatifida, S. baetica, S. reverchonii, S. angustifolia, S. laciniata var.calcitrapifolia and var.subulata (2n = 14) are new. Diploid cytotypes with 2n = 14 and 2n = 12 prevail, andS. hispanica var.crispatula is the only taxon which exhibits autopolyploidy (2n = 14, 28). x = 7 is considered to be the base chromosome number within the genus, with x = 6 being derived from it by translocation. This and detailed karyotype analyses allow to group the Iberian Peninsula species ofScorzonera into three groups.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Effect of temperature on the photoreduction of centres A and B in Photosystem I,and the kinetics of recombination

When spinach Photosystem I particles, frozen in the dark with ascorbate, are illuminated at low temperatures, one electron is transferred from P-700 to either iron-sulphur centre A or B. It was found that the proportion of centre A or B reduced depended on the temperature of illumination. At 25 K, reduction of centre A, as detected by ESR spectroscopy, was strongly preferred. At higher temperatures, at about 150K, there was an increased proportion of reduced centre B. Reduction of B was more strongly preferred in particles frozen in 50% glycerol. The kinetics of dark reoxidation of A^? and B^? at various temperatures were followed by observing the radical signal of P-700⁺, and also by periodically cooling to 25 K to measure the ESR spectra of the iron-sulphur centres. The recombination of A^? and P-700⁺ occurred at lower temperatures than that at of B^?; at 150–200 K, centre B was the more stable electron trap. Dark reoxidation of both centres was more rapid in samples that were illuminated at 25 K than in samples illuminated at 150–215 K. In no case was net electron transfer between centres A and B observed. Differences in g values of the ESR spectra in particles illuminated at 25 and 200 K indicate that the iron-sulphur centres are in altered conformational states. It is concluded firstly that, in the frozen state, the rates of dark electron transfer decrease in the sequence A^? → P-700⁺ > B^? → P-700⁺ > B^? → A; secondly, that when centres A or B are photoreduced, a temperature-dependent conformational change takes place which slows down the rate of recombination with P-700⁺.     

Thermodynamics of base interaction in (A)n and (A.U)n

Using precision scanning microcalorimetry we studied (A)_n and (A·U)_n melting in highly diluted solutions (0.3 to 5.0 mm) with different Na⁺ activity. This permitted us to determine directly the thermodynamic functions of stacking interaction in (A)_n and base-pairing in (A·U)_n. For (A-A) stacking at (A)_n melting temperature we obtained ΔH^(A)n_m = 12.6 kJ mol^?1; ΔS^(A)n_m = 41 J K^?1 mol^?1. For A·U base-pairing at a standard temperature of 298 K and 0.1 m-Na⁺ we have: ΔH^(A·U) = 34 kJ mol^?1; ΔS^(A·U) = 102 J K^?1 mol^?1ΔG^(A·U) = ?3.5 kJ mol^?1.     

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle

Ca²⁺-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca²⁺ inhibited cardiac RyRs with an average K _m = 15 mm, skeletal RyRs with K _m = 0.7 mm and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca²⁺ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [³H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca²⁺-inhibition in cardiac RyRs (Chu, Fill, Stefani &; Entman (1993) J. Membrane Biol. 135, 49–59). Ryanodine prevented Ca²⁺ from inhibiting either cardiac or skeletal RyRs. Ca²⁺-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mm Cs⁺, but not by 500 mm K⁺, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca²⁺-activation. Ca²⁺-inhibition in skeletal RyRs was retained in the presence of 500 mm Cs⁺. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mm Cs⁺ in the solutions fail to demonstrate Ca²⁺-inhibition, while Ca²⁺-inhibition of Ca²⁺ release is observed in vesicle studies where K⁺ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca²⁺-inhibition in skeletal RyRs and cardiac RyRs, with different Ca²⁺ affinities for inhibition. We conclude that differences in the Ca²⁺-inhibition in cardiac and skeletal channels depends on their Ca²⁺ binding properties.     

Karyological and taxonomic observations onDracocephalum foetidum Bunge andKoenigia islandica L.

Chromosome numbers are reported for two Mongolian species,Dracocephalum foetidum Bunge (2n=12) andKoenigia islandica L. (2n=14). The relationship ofD. foetidum toD. moldavica L. (2n=10) and some patterns of phenotypic variation inK. islandica are briefly discussed. The following new combinations are proposed:K. cyanadra (Diels) Měsí?ek etSoják,K. forrestii (Diels) Měsí?ek etSoják,K. hubertii (Lingelsh.) Měsí?ek etSoják, andK. nummularifolia (Meisn.) Měsí?ek etSoják.     

An Inactivating Inward-rectifying K Current Present in Prolactin Cells from the Pituitary of Lactating Rats

Primary cultures containing a high percentage of lactotrophs were obtained by dissociating the pituitary of rats following 14–18 days of lactation. Lactotrophs with a distinctive appearance were recorded after 1–35 days in vitro and identified by immunocytochemical staining for prolactin. Whole-cell voltage clamp measurements in isotonic KCl solution from a holding potential of −40 mV revealed the presence of inward-rectifying K currents with a time-dependent, Na⁺-independent inactivation at potentials negative to −60 mV. The time for complete inactivation was strikingly different between lactotrophs, varying between 1 sec and more than 5 sec at −120 mV, and was not related to time in culture. The reversal potential shifted 59 mV (25°C) for a tenfold change in external K⁺ concentration, demonstrating the selectivity of the channel for K⁺ over Na⁺. The inward-rectifying K current was blocked by 5 mm Ba²⁺ and partially blocked by 10 mm TEA. Chloramine-T (1 and 2 mm) produced a total block of the inward-rectifying K current in lactotrophs. Thyrotropin-releasing hormone (500 nm) significantly reduced the inward-rectifying K current in about half of the lactotrophs. This current is similar to the inward-rectifying K current previously characterized in clonal somatomammotrophic pituitary cells (GH₃B₆). The variability of the rate of inactivation of this current in lactotrophs and its responsiveness to TRH is discussed. Received: 28 September 1995/Revised: 11 December 1995     

Mechanisms of cell volume regulation in the proximal segment of the Malpighian tubule of Rhodnius neglectus

The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using videooptic techniques. When the medium osmolality was increased with addition of 100 mm mannitol the cells shrank to a minimum of 16.84±2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K⁺ or Cl^? or HCO ₃ ^? by Na⁺, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na⁺ by tetramethylammonium (TMA⁺) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10^?4 m, anthracene-9-COOH 5×10^?4 m, furosemide 5×10^?4 m or ethacrynic acid 5×10^?5 m, also abolished RVI. On the other hand, addition of either Ba²⁺ 10^?3 m, SITS 5× 10^?4 m, ouabain 10^?3 m or vanadate 10^?3 m, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mm or verapamil 10^?6 m, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mm induced a cell swelling to a maximum of 33.11+1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na⁺, K⁺, Cl^?, Ca²⁺ and HCO ₃ ^? transport pathways and a ouabain-insensitive ATPase stimulated by Na⁺. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq e Financiadora de Projetos e Pesquisas-FINEP.     

Cytomolecular discrimination of the A<Superscript>m</Superscript> chromosomes of <Emphasis Type="Italic">Triticum monococcum</Emphasis> and the A chromosomes of <Emphasis Type="Italic">Triticum aestivum</Emphasis> using microsatellite DNA repeats

The cytomolecular discrimination of the A^m- and A-genome chromosomes facilitates the selection of wheat-Triticum monococcum introgression lines. Fluorescence in situ hybridisation (FISH) with the commonly used DNA probes Afa family, 18S rDNA and pSc119.2 showed that the more complex hybridisation pattern obtained in T. monococcum relative to bread wheat made it possible to differentiate the A^m and A chromosomes within homoeologous groups 1, 4 and 5. In order to provide additional chromosomal landmarks to discriminate the A^m and A chromosomes, the microsatellite repeats (GAA)_n, (CAG)_n, (CAC)_n, (AAC)_n, (AGG)_n and (ACT)_n were tested as FISH probes. These showed that T. monococcum chromosomes have fewer, generally weaker, simple sequence repeat (SSR) signals than the A-genome chromosomes of hexaploid wheat. A differential hybridisation pattern was observed on 6A^m and 6A chromosomes with all the SSR probes tested except for the (ACT)_n probe. The 2A^m and 2A chromosomes were differentiated by the signals given by the (GAA)_n, (CAG)_n and (AAC)_n repeats, while only (GAA)_n discriminated the chromosomes 3A^m and 3A. Chromosomes 7A^m and 7A could be differentiated by the lack of (GAA)_n and (AGG)_n signals on 7A. As potential landmarks for identifying the A^m chromosomes, SSR repeats will facilitate the introgression of T. monococcum chromatin into wheat.     

Nitrilotris(methylenephosphonates) in aqueous solution and solid state - dilatometric, potentiometric and NMR investigations

Dissociation and alkali complex formation equilibria of nitrilotris(methylenephosphonic acid) (NTMP, H₆L) have been studied by dilatometric, potentiometric and ³¹P NMR-controlled titrations. Dilatometry indicated the formation of alkali complexes ML (M=Li, Na, K, Rb, Cs) at high pH with a stability decreasing from Li to Cs. An efficient combination of potentiometric and NMR methods confirmed two types of alkali metal complexes MHL and ML. Stability constants for the equilibria following M⁺ + HL⁵⁻ ? MHL⁴⁻ and M⁺ + L⁶⁻ ? ML⁵⁻, respectively, were determined: logK_NaHL=1.08(0.07), logK_KHL=0.86(0.08), logK_NaL=2.24(0.03). Systematic errors are introduced by using alkali metal hydroxides as titrants for routine potentiometric determinations of dissociation constants pK_a5^app and pK_a6^app. Correction formulae were derived to convert actual dissociation constants pK_a into apparent dissociation constants pK_a^app (or vice versa). The actual dissociation constants were found: pK_a5(H₂L⁴⁻ ? H⁺ + HL⁵⁻)=7.47(0.03) and pK_a6(HL⁵⁻ ? H⁺ + L⁶⁻)=14.1(0.1). The anisotropy of ³¹P chemical shifts of salts M_nH_6 − nL (M=Li, Na, n=0-5) is more sensitive towards titration (n) than isotropic solution state chemical shifts.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

Syntheses and crystal structures of three diorganoltin(IV) macrocycles

Three novel macrocyclic diorganotin(IV) compounds of the type: {[R₁₀(SnO)₃(SnOH)₂]H_nXO_m}₂ · L (n=1, m=4, R=PhCH₂, X=P, L=0, 1; n=0, m=4, R=PhCH₂, X=S, L=4H₂O, 2; n=0, m=3, R=n-Bu, X=N, L=0, 3) were synthesized by the reaction of (PhCH₂)₂SnCl₂ with Na₂H_nXO₄ (n=1, X=P; n=0, X=S) or (n-Bu)₂SnCl₂ with NaNO₃. All the compounds 1, 2 and 3 are characterized by elemental, IR and X-ray diffraction analyses. X-ray data reveal that a macrocyclic structure with two centrosymmetric ladders of hydrolysis exists in the crystals of the three compounds. The geometry about each tin atom involved is trigonal bipyramidal.