Differential methylation at the 5′ and the 3′ CCGG sites flanking the X chromosomal hypervariable DXS255 locus

Summary The degree of methylation at the 5 and 3 CCGG sequences flanking the variable number of tandem repeat (VNTR) region of the DXS255 locus at Xp11.22 was analysed separately in several haematopoietic cell lineages. The 5 CCGG site on active chromosomes was found to be completely methylated in B and T lymphocytes and granulocytes. Methylation of the 5 site on inactive X chromosomes differed between females (0%–60%), but was consistent in different cell lineages obtained from individual females. In contrast, methylation at the 3 CCGG site on active chromosomes was found to vary in B lymphocytes (40%–100%), whereas complete methylation was found in T lymphocytes and granulocytes. The extent of methylation on inactive X chromosomes was found to differ significantly between B lymphocytes (17%), T lymphocytes (54%) and granulocytes (82%). Thus, methylation at the 5 CCGG site seems to be primarily related to the status of X chromosome inactivation, whereas methylation at the 3 CCGG site is mainly subject to cell-lineage-specific influences.     

Unbalanced translocation between chromosomes 2 and 7 with de novo deletion of band 35 on the long arm of chromosme 2

Summary Clinical and cytogenetical findings are described in an infant with a de novo deletion of the long arm of chromosome 2. The boy's karyotype is 46,XY, rec(2)delq,t(2;7) (2pter2q34::7p217pter) (7qter7p21::2q362qter). He showed developmental retardation, low-set ears, micrognathia, short neck, abundant skin of the neck, tetralogy of Fallot, bipartite labialike scrotum, clitorislike penis, cryptorchism, and deformities of the hands and feet.     

Gene expression, cellular localization, and enzymatic activity of diacylglycerol kinase isozymes in rat ovary and placenta

Female reproductive organs show remarkable cyclic changes in morphology and function in response to a combination of hormones. Evidence has accumulated suggesting that phosphoinositide turnover and the consequent diacylglycerol (DG) protein kinase C (PKC) pathway are intimately involved in these mechanisms. The present study has been performed to investigate the gene expression, cellular localization, and enzymatic activity of the DG kinase (DGK) isozymes that control the DG-PKC pathway. Gene expression for DGK, -, -, and - was detected in the ovary and placenta. Intense expression signals for DGK and - were observed in the theca cells and moderate signals in the interstitium and corpora lutea of the ovary. On the other hand, signals for DGK were seen more intensely in granulosa cells. In the placenta, signals for DGK and - were observed in the junctional zone, whereas those for DGK were detected in the labyrinthine zone. At higher magnification, the signals for DGK were mainly discerned in giant cytotrophoblasts, and those for DGK were found in small cytotrophoblasts of the junctional zone. DGK signals were observed in all cellular components of the labyrinthine zone, including mesenchyme, trabecular trophoblasts, and cytotrophoblasts. DGK signals were detected in the junctional zone on day 13 and 15 of pregnancy and were diffusely distributed both in the labyrinthine and junctional zones at later stages. The present study reveals distinct patterns of mRNA localization for DGK isozymes in the rat ovary and placenta, suggesting that each isozyme plays a unique role in distinct cell types in these organs.This work was supported by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, the Uehara Memorial Foundation, the ONO Medical Research Foundation, the Ciba-Geigy Foundation (Japan) for the Promotion of Science, the Kato Memorial Bioscience Foundation, and the Yamagata Health Support Foundation (to K.G.).     

Non-PTS uptake and subsequent metabolism of glucose in Pediococcus halophilus as demonstrated with a double mutant defective in phosphoenolpyruvate:mannose phosphotransferase system and in phosphofructokinase

Pediococcus halophilus possesses phosphoenolpyruvate:mannose phosphotransferase system (man:PTS) as a main glucose transporter. A man:PTS defective (man:PTS^d) strain X-160 could, however, utilize glucose. A possible glucose-transport mechanism other than PTS was studied with the strain X-160 and its derivative, man:PTS^d phosphofructokinase defective (PFK^–) strain M-13. Glucose uptake by X-160 at pH 5.5 was inhibited by any of carbonylcyanide m-chlorophenylhydrazone, nigericin, N,N-dicyclohexylcarbodiimide, or iodoacetic acid. The double mutant M-13 could still transport glucose and accumulated intracellularly a large amount of hexose-phosphates (ca. 8 mM glucose 6-phosphate and ca. 2 mM fructose 6-phosphate). Protonophores also inhibited the glucose transport at pH 5.5, as determined by the amounts of accumulated hexose-phosphates (< 4 mM). These showed involvement of proton motive force (P) in the non-PTS glucose transport. It was concluded that the non-PTS glucose transporter operated in concert with hexokinase or glucokinase for the metabolism of glucose in the man:PTS^d strain.Abbreviations BM basal medium - BM-G basal medium containing glucose - CM complex medium - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - P proton motive force - pH transmembrane pH gradient - transmembrane electrical potential difference - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PIPES piperazine-N,N-bis(-ethanesulfonic acid) - MES 4-morpholineethanesulfonic acid - G-6-P glucose 6-phosphate - F-6-P fructose 6-phosphate - FDP fructose 1,6-bisphosphate - EMP Embden-Meyerhof-Parnas pathway - PFK phosphofructokinase - GK glucokinase - HK hexokinase - IAA iodoacetic acid - II^man enzyme II component of man:PTS     

Elucidation of Structure of Lipid A from the Marine Gram-Negative Bacterium Pseudoalteromonas haloplanktis ATCC 14393T

The chemical structure of lipid A, from the marine -proteobacterium Pseudoalteromonas haloplanktis 14393, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be -1,6-glucosaminobiose 1,4-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3 and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2, respectively.     

Backbone dynamics of the cytotoxic ribonuclease alpha-sarcin by 15N NMR relaxation methods

The cytotoxic ribonuclease -sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a strictly conserved rRNA loop. In order to gain insights on the molecular basis of its highly specific activity, we have previously determined its solution structure and studied its electrostatics properties. Here, we complement those studies by analysing the backbone dynamics of -sarcin through measurement of longitudinal relaxation rates R₁, off resonance rotating frame relaxation rates R₁, and the ¹⁵N¹HNOE of the backbone amide ¹⁵N nuclei at two different magnetic field strengths (11.7 and 17.6 T). The two sets of relaxation parameters have been analysed in terms of the reduced spectral density mapping formalism, as well as by the model-free approach. -Sarcin behaves as an axial symmetric rotor of the prolate type (D/D=1.16 ± 0.02) which tumbles with a correlation time _m of 7.54 ± 0.02 ns. The rotational diffusion properties have been also independently evaluated by hydrodynamic calculations and are in good agreement with the experimental results. The analysis of the internal dynamics reveals that -sarcin is composed of a rigid hydrophobic core and some exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the s to ms time scale are less abundant and in some cases can be assigned to specific motional processes. All dynamic data are discussed in relation to the role of some particular residues of -sarcin in the process of recognition of its ribosomal target.     

The mechanism of lactate transport in human erythrocytes

Summary Lactate accumulates in human erythrocytes stored at 4°C in the presence of glucose. Efflux of lactate exhibits an activation energy of 22 kcal/mole and is markedly stimulated with increasing medium pH. Lactate influx into erythrocytes that were depleted of intracellular lactate by incubation at 37° at pH 8.0 was stimulated by decreasing medium pH. Under appropriate conditions the pH-dependent lactate flux was insensitive to 4-acetamido-4-isothiocyano-2,2-disulfonic stilbene or 4,4-diisothiocyano-2,2-disulfonic stilbene, inhibitors of the inorganic anion channel, while, e.g., inorganic phosphate transport was fully sensitive. These experiments as well as measurements of H⁺ movements associated with lactate fluxes demonstrate that lactate transport takes place via a specific monocarboxylate transporter (distinct from the inorganic ion channel) by a H⁺-lactate symport mechanism.     

Internal motions of apo-neocarzinostatin as studied by 13C NMR methine relaxation at natural abundance

Summary Dynamics of the backbone and some side chains of apo-neocarzinostatin, a 10.7 kDa carrier protein, have been studied from ¹³C relaxation rates R1, R2 and steady-state ¹³C-{¹H} NOEs, measured at natural abundance. Relaxation data were obtained for 79 nonoverlapping C resonances and for 11 threonine C single resonances. Except for three C relaxation rates, all data were analysed from a simple two-parameter spectral density function using the model-free approach of Lipari and Szabo. The corresponding C–H fragments exhibit fast (_e < 40 ps) restricted libration motions (S²=0.73 to 0.95). Global examination of the microdynamical parameters S² and _e along the amino acid sequence gives no immediate correlation with structural elements. However, different trends for the three loops involved in the binding site are revealed. The -ribbon comprising residues 37 to 47 is spatially restricted, with relatively large _e values in its hairpin region. The other -ribbon (residues 72 to 87) and the large disordered loop ranging between residues 97–107 experience small-amplitude motions on a much faster (picosecond) time scale. The two N-terminal residues, Ala¹ and Ala², and the C-terminal residue Asn¹¹³, exhibit an additional slow motion on a subnanosecond time scale (400–500 ps). Similarly, the relaxation data for eight threonine side-chain C must be interpreted in terms of a three-parameter spectral density function. They exhibit slower motions, on the nanosecond time scale (500–3000 ps). Three threonine (Thr⁶⁵, Thr⁶⁸, Thr⁸¹) side chains do not display a slow component, but an exchange contribution to the observed transverse relaxation rate R2 could not be excluded at these sites. The microdynamical parameters (S², _e and R2_ex) or (S _infslow ^sup2 , S _inffast ^sup2 and _slow) were obtained from a straightforward solution of the equations describing the relaxation data. They were calculated assuming an overall isotropic rotational correlation time _e for the protein of 5.7 ns, determined using standard procedures from R2/R1 ratios. However, it is shown that the product (1–S²)× _e is nearly independent of _e for residues not exhibiting slow motions on the nanosecond time scale. In addition, this parameter very closely follows the heteronuclear NOEs, which therefore could be good indices for local fast motions on the picosecond time scale.     

Enzymatic semisynthesis of aprotinin homologues mutated in P′ positions

The replacement of amino acids in the P₁ and P₂ position of aprotinin, the bovine pancreatic trypsin inhibitor, is described. Using the modified inhibitor as starting material, with the hydrolyzed reactive-site peptide bond Lys¹⁵-Ala¹⁶, the residues P₁ (Ala¹⁶) and P₂ (Arg¹⁷) were split off by the action of aminopeptidase K. Incorporation of suitable dipeptides containing a basic residue (Lys or Arg) in the C-terminal position was carried out in a one pot reaction involving trypsin-catalyzed coupling. In this way, the native fragment Ala¹⁶-Arg¹⁷ was reintroduced and also replaced by Gly-Arg, Ala-Lys, and Leu-Arg yielding intact inhibitor molecules. The mechanism for incorporation of dipeptides was investigated by treating the aprotinin derivative with the Arg¹⁷-Ile¹⁸ peptide bond hydrolyzed with trypsin under proteosynthetic conditions. We established that only inhibitor molecules cleaved between Lys¹⁵ and Xaa¹⁶ are intermediates leading to the desired products. The inhibitory properties of the new aprotinin homologues were tested, and the significance of the P₁ residue for the inhibition of trypsin, kallikrein, and chymotrypsin was deduced.     

High-level Expression of Maize <Emphasis Type="Italic">γ</Emphasis>-zein Protein in Transgenic Soybean (<Emphasis Type="Italic">Glycine max</Emphasis>)

Primers were developed for 118 microsatellites isolated from grape (Vitis vinifera) genomic libraries enriched for (AC)n repeats. Only one microsatellite sequence matched other grape SSR-sequences in the GeneBank database. Genotyping was carried out in the parental lines and four offspring of two pseudo-test-cross populations, Cabernet Sauvignon x Seyval and Chardonnay x Bianca, and a further six other grape genotypes (V. vinifera Sultanina, Merlot, Syrah, Müller-Thurgau, Vitis Regent and V. riparia Gloire de Montpellier). A total of 108 microsatellites showed easily scorable alleles and 100 of them segregated according to a configuration suitable for mapping in either cross. A further 8 SSRs, although unsuitable for mapping in those crosses, showed polymorphism in the other genotypes tested. This set of markers was used, along with 75 microsatellites of other repeat-types, to fingerprint 46 offspring of the cross Chardonnay x Bianca. For each full-sib, individual heterozygosity and distance in repeat units between pairs of alleles at each locus (mean d²) were calculated as a tool for predicting highly outbred recombinant individuals. Six microsatellites with segregation ratios significantly distorted towards the lack of homozygous sibs were identified and mapped to linkage groups LG 3 and LG 5. Estimation of heterozygosity at genome-wide level and genotyping at loci for which homozygous sibs are discriminated against are discussed for marker-assisted background selection in outcrossing grapevines.     

Sources and inheritance of resistance to leaf curl virus in Lycopersicon

Summary One hundred and twenty-two varieties, lines and wild accessions of Lycopersicon were screened under three different regimes during the autumn/winter season of 1982–83 and 1983–84 for resistance to tomato leaf curl virus (TLCV). L. hirsutum f. glabratum (B6013) and L. hirsutum f. typicum (A1904) proved to be highly resistant to TLCV in all three environments. Various accessions of L. peruvianum were also highly resistant. L. pimpinellifolium (A1921) exhibited no TLCV symptoms within 90 days. Of the cultivated varieties, Acc 99 exhibited the minimim score for susceptibility; AC 142, Collection No. 2, Kalyanpur Angurlata and HS 101 had a low rating for virus incidence. The inheritance of resistance was studied in the interspecific crosses between a TLCV resistant line of L. pimpinellifolium (A1921) and five (HS 101, HS 102, HS 110, Pusa Ruby and Punjab Chhuhara) susceptible cultivars of L. esculentum. Parents, F₁, F₂ and backcross progenies were artificially inoculated with local strains of TLCV using vector the viruliferious whitefly, Bemisia tabaci (Genn.). Data indicated that the resistance of L. pimpinellifolium (A 1921) is monogenic and incompletely dominant over susceptibility.     

Effects of midazolam on the contraction and relaxation of segments of thoracic aorta stripped of endothelium and stimulated by adrenaline – experimental study in rabbits

To evaluate the effects of midazolam on the angiokinesis of segments of rabbits' thoracic aorta stripped of endothelium and stimulated by adrenaline.Two groups of aortic rings removed from albinic rabbits anesthetized with thiopental were used (Group I – 6 animals; Group II – 12 animals), stripped of endothelium, studied in an organ chamber, perfused by Krebs-Henseleit solution. The groups were stimulated by adrenaline, recording the maximum contraction and dT/dt at 12, 36, 60 and 120. When the plateau phase was reached, the vessel was washed with perfusion solution, recording relaxation at 2, 4 and 6. When the base values were reached, Group I underwent a new adrenergic stimulus; and Group II was stimulated with midazolam and then with adrenaline, and the same values were recorded. T test was applied as a statistical analysis when two variables were studied. When studying more than two variables the Anova test was used, supplemented by the Tuckey test.Group I did not show any significant difference between the two stimuli. Group II – the midazolam significantly reduced the maximum contraction induced by adrenaline (83.01 ± 4.11%) (p < 0.01). The dT/dt was reduced at 12 (57.06 ± 8.47%), and also at 36 (70.59 ± 5.26%). There was no significance at 60 and 120 (p < 0.01).The relaxation increased significantly at all measurements – at 2-adrenaline 39.31 ± 9.60%; adrenaline/midazolam: 44.06 ± 9.62% (p < 0.05). At 4-adrenaline: 53.08 ± 8.3%; adrenaline/midazolam: 61.68 ± 8.50% (p < 0.01). At 6-adrenaline: 76.26 ± 5.45%; adrenaline/midazolam: 84.20 ± 7.96% (p < 0.01).Midazolam significantly reduced the maximum contraction obtained by the adrenergic stimulus as well as the dT/dt in the initial phases of contraction. The relaxation speed also increased.     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Occurrence of airborne Cladosporium and Alternaria spores in Southern and Central Poland in 1995–1996

The concentration of airborne spores of Cladosporium and Alternaria has been investigated at five monitoring stations situated in cities from the foot of the Tatra Mountains to central Poland along a south-north transect (Zakopane, Kraków, Ostrowiec witokrzyski, Warszawa, Pozna) i.e. from a height of 900 m to 80 m above sea level. The aerobiological monitoring of fungal spores was performed by means of five Burkard volumetric spore traps.Cladosporium spores were dominant at all the stations. The highest Cladosporium and Alternaria spore concentrations were observed at all the sites in July and August, except at Warszawa in both years and at Pozna in 1995 where the maximum of Cladosporium spores occurred in June and July, and at Ostrowiec witokrzyski in 1995 where the maximum was found in July, August and September.Fungal spore concentrations in Zakopane and Kraków were significantly lower than those in Ostrowiec witokrzyski, Warszawa and Pozna and periods of abundant Cladosporium spore occurrence were different in these two groups of monitoring stations.     

Mutagenic effectiveness and efficiency of EMS,DES and gamma-rays in rice

Summary Data on chlorophyll mutation frequency after treatment with EMS, DES and gamma-rays and sequential administration of gamma-rays and the two alkylating agents in three varieties of rice have been used to work out quantitatively the effectiveness and efficiency of each mutagen and combination treatment. For effectiveness, the order is EMS > DES and for efficiency it is EMS > DES > gamma-rays. In some sequential treatments (Gamma-rays + DES in IR8 and Basmati; DES + gamma-rays in IR8 and Jhona; Gamma-rays + EMS in IR8 and Basmati; and EMS + gamma-rays in IR8, Jhona and Basmati) mutation frequency is more than additive (synergistic) but these treatments are decisively less efficient because of their relatively high injurious effects in the M₁. generation. EMS induces more albinas than gamma-rays do. The mutational spectrum patterns induced by gamma-rays and DES are alike. In general, combination treatments tend to increase the frequency of albinas over other types of chlorophyll mutants.     

Conformation of the circular dumbbell d〈pCGC-TT-GCG-TT〉: Structure determination and molecular dynamics

Summary The circular DNA decamer 5-dpCGC-TT-GCG-TT-3 was studied in solution by means of NMR spectroscopy and molecular dynamics in H₂O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5-GTTC-3 loop with only one remaining (solvent-accessible) hydrogen bond between NH of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5-CTTG-3 loop remains stable. The two conformers occur in slow equilibrium (rate constant 2–20 s^–1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (³J_HH, ³J_HP, ³J_CP) gave detailed information about the local geometry around backbone torsion angles , , and , revealing a relatively high flexibility of the 5-GTTC-3 loop. The values of the coupling constants are virtually temperature-independent. Weakly constrained molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over r^–3 to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S² factors and r^–6 averaging of the r^–3-averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5-GTTC-3 loop. The difference in stability of the 5-CTTG-3 and 5-GTTC-3 loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle .     

Viability and activity in readily culturable bacteria: a review and discussion of the practical issues

In microbiology the terms viability and culturability are often equated. However, in recent years the apparently self-contradictory expression viable-but-nonculturable (VBNC) has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms viability and culturability in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces VBNC with expressions that are internally consistent.     

On morphological variation in Keratella cochlearis populations from Holstein lakes (Northern Germany)

Keratella cochlearis occurs in many Holstein lakes (northern Germany) as three well defined and separated forms: cochlearis, hispida, and tecta, each showing very little variation between the lakes. The present data show that the tecta form did not originate from a Lauterborn cycle.