Variation patterns within the genusCarex L. sect.Digitatae (Fries) Christ in Bulgaria

Seventeen natural Bulgarian populations of the species of the genusCarex sect.Digitatae were studied morphologically and karyologically. Stepwise discriminant analysis was used as a multivariate test for the separation of groups of populations from different taxa and to find a subset of characters contributing most for this separation. The following taxa were found in Bulgaria:C. humilis Leysser var.humilis; C. humilis var.longifolia Stoeva etPopova, var. n.;C. digitata L.;C. ornithopoda Willd. subsp.bulgarica (Vel.) Stoeva etPopova, comb. n. Aneuploid series of some chromosome numbers were found inC. digitata — 2n=48, 50, 52, 54, 56 and inC. ornithopoda subsp.bulgarica?2n=52, 53, 54, 56.C. humilis has 2n=36 and a much more heterogeneous karyotype than that of the above taxa. The values of the Euclidean distance between the populations vary within similar limits in both speciesC. humilis andC. digitata; this is not in accordance with the karyological results. The within and between population morphological variations were compared in each species. The first dominates in overall variation, which is probably connected with the very large populations and their mosaic space structure.     

The role of the C terminus and Na+/H+ exchanger regulatory factor in the functional expression of cystic fibrosis transmembrane conductance regulator in nonpolarized cells and epithelia

The conserved C-terminal peptide motif (1476DTRL) of the cystic fibrosis transmembrane conductance regulator (CFTR) ensures high affinity binding to different PSD-95/Disc-large/zonula occludens-1 (PDZ) domain-containing molecules, including the Na+/H+ exchanger regulatory factor (NHERF)/ezrin-radixin-moesin-binding phosphoprotein of 50 kDa. The physiological relevance of NHERF binding to CFTR is not fully understood. Individuals with mutations resulting in premature termination of CFTR (S1455X or Delta26 CFTR) have moderately elevated sweat Cl- concentration, without an obvious lung and pancreatic phenotype, implying that the CFTR function is largely preserved. Surprisingly, when expressed heterologously, the Delta26 mutation was reported to abrogate channel activity by destabilizing the protein at the apical domain and inducing its accumulation at the basolateral membrane (Moyer, B., Denton, J., Karlson, K., Reynolds, D., Wang, S., Mickle, J., Milewski, M., Cutting, G., Guggino, W., Li, M., and Stanton, B. (1999) J. Clin. Invest. 104, 1353-1361). The goals of this study were to resolve the contrasting clinical and cellular phenotype of the Delta26 CFTR mutation and evaluate the role of NHERF in the functional expression of CFTR at the plasma membrane. Complex formation between CFTR and NHERF was disrupted by C-terminal deletions, C-terminal epitope tag attachments, or overexpression of a dominant negative NHERF mutant. These perturbations did not alter CFTR expression, metabolic stability, or function in nonpolarized cells. Likewise, inhibition of NHERF binding had no discernible effect on the apical localization of CFTR in polarized tracheal, pancreatic, intestinal, and kidney epithelia and did not influence the metabolic stability or the cAMP-dependent protein kinase-activated chloride channel conductance in polarized pancreatic epithelia. On the other hand, electrophysiological studies demonstrated that NHERF is able to stimulate the cAMP-dependent protein kinase-phosphorylated CFTR channel activity in intact cells. These results help to reconcile the discordant genotype-phenotype relationship in individuals with C-terminal truncations and indicate that apical localization of CFTR involves sorting signals other than the C-terminal 26 amino acid residues and the PDZ-binding motif in differentiated epithelia.     

Chemical, photochemical and spectroscopic characterization of an alkaline proteinase from Bacillus subtilis variant DY

Circular-dichroism and fluorescence studies indicate that the 5-dimethylaminonaphthalene-1-sulphonyl and phenylmethanesulphonyl derivatives of subtilisin DY have three-dimensional structure closely similar to that of native enzyme. The single tryptophan residue is largely accessible to the aqueous solvent, and is not directly involved in the enzyme-substrate interactions, since its photochemical modification causes only a partial inhibition of the enzyme activity. It appears very likely that the location of the single tryptophan residue in the three-dimensional structure of subtilisin DY is similar to that of the single tryptophan residue in subtilisin Carlsberg. Fluorescence-quenching experiments further indicate that the 14 tyrosine residues are also largely accessible to the aqueous solvent, and probably interact with hydrated peptide carbonyl groups. The charge environment for tryptophan and tyrosine residues in subtilisin DY, as deduced by quenching experiments with ionic species, is also discussed. In general, subtilisin DY displays strong similarities to subtilisin Carlsberg, as suggested by a comparative analysis of the amino acid composition and fluorescence properties.     

Effects of pH and urea on the conformational properties of subtilisin DY

Subtilisin DY is very resistant to the denaturing action of urea: the conformational properties are not affected up to 4.5 M-urea, and even in the presence of 8 M-urea there is only a slow loss of ordered structure and caseinolytic activity. C.d. and fluorescence-emission studies also show that this proteinase is stable in the 5.5-10.0 pH range, whereas below pH 5.5 a sharp denaturation occurs that is complete at pH 4.5. Protein denaturation leads to a change of the emission quantum yield; in particular, in the native protein, indole fluorescence is quenched by some amino groups. Moreover, subtilisin DY possesses two classes of tyrosine residues: one class of exposed residues titrates normally, with pKapp. = 10.24, whereas one class of partially buried or hydrogen-bonded residues ionizes with pKapp. = 11.58. In general, such conformational properties resemble those of other subtilisins. However, some differences occur: e.g., subtilisin DY is less stable at acidic pH values and its tyrosine residues are more accessible to the solvent. Such differences are probably due to small variations of the three-dimensional structure; e.g., subtilisin DY has a slightly lower alpha-helix content.     

Analysis of differences in photosynthetic nitrogen use efficiency of alpine and lowland Poa species

This study investigates factors determining variation in photosynthetic nitrogen use efficiency (φ_N) in seven slow- and fast-growing Poa species from altitudinally contrasting sites. The species and their environmental origin were (in order of increasing relative growth rate): two alpine (Poa fawcettiae and P. costiniana), one sub-alpine (P. alpina) and three temperate lowland perennials (P. pratensis, P. compressa and P. trivialis), as well as one temperate lowland annual (P. annua). Plants were grown hydroponically under identical conditions with free access to nutrients in a growth room. Photosynthesis per unit leaf area measured at growth irradiance (500 μmol m⁻² s⁻¹) was slightly higher in the slow-growing alpine species. At saturating light intensities, photosynthesis was considerably higher in the alpine species than in the lowland species. Carboxylation capacity and Rubisco content per unit leaf area were also greater in the alpine species. Despite variation between the species, the in vivo specific activity of Rubisco showed little relationship to relative growth rate or photosynthetic rate. Both at light saturation and at the growth irradiance, φ_N was lowest in the slow-growing alpine species P. fawcettiae, P. costiniana and P. alpina, and highest in the fast-growing P. compressa and P. annua. The proportion of leaf nitrogen that was allocated to photosynthetic capacity and the in vivo catalytic constant of Rubisco accounted for most of the variation in φ_N at light saturation. Minor variations in intercellular CO₂ partial pressure also contributed to some extent to the variations in φ_N at light saturation. The low φ_N values at growth irradiance exhibited by the alpine species were additionally due to a lower percentage utilisation of their high photosynthetic capacity compared to the lowland species. Received: 28 May 1998 / Accepted: 28 March 1999     

Mental retardation in childhood: clinical and diagnostic profile in 100 children

Mental retardation (MR) is present in 2-3% of individuals in the general population, either as an isolated finding, or as part of an underlying disorder. It is the reason for a substantial part of referrals of patients and families to the pediatric genetic counseling units. MR may be genetically determined, or due to environmental (including perinatal) influences. Despite the thorough examination, physicians often are unable to define the etiology, in at least 30-50% of cases. This report presents the experience with 100 consecutive children with mental retardation, admitted to the Section of clinical genetics of the University pediatric hospital in Sofia over one year period. According to the routinely used classification, the patients' disease states have been subgrouped into genetic (35), multifactorial (3), environmental (3), and of unknown etiology (59). The research protocol included careful dysmorphologic, neurologic and developmental assessment, as well as cytogenetic, biochemical and molecular genetic testing. The rate of the diagnostic yield of the whole group was 41%. The discussion includes clinical examples and literature reports.     

Development of a BRET2 screening assay using beta-arrestin 2 mutants

This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and beta-arrestin 2 (beta-arr2), measured by the bioluminescence resonance energy transfer (BRET(2)) technology. Both class A (beta(2)-adrenergic receptor [beta(2)-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET(2) signal can be enhanced by using (1) beta-arr2 phosphorylation-independent mutant (beta-arr2 R169E) and (2) beta-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (beta-arr2 R393E, R395E and beta-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET(2) signal when comparing results obtained with wild-type (wt) and mutant beta-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET(2) signal was observed with beta-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these beta-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET(2) signal. The beta-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET(2)-based agonist and antagonist screening assays.     

Sesquiterpene lactones from Anthemis carpatica Willd

Four new eudesmanolides and two new guaianolides were isolated from the aerial parts of Anthemis carpatica Willd. and their structures elucidated by spectral methods. In addition, seven known sesquiterpene lactones were identified.     

Baker's yeast production from molasses/cheese whey mixtures

Partial substitution of sugarcane molasses by cheese whey in the fed-batch production of baker's yeast was evaluated. A sugar feeding profile based in a commercial process and different modes of addition of -galactosidase were used. Molasses substitution of 46%, in terms of sugar fed to the bioreactor, was reached and no significant differences in biomass volumetric productivity, by-products yields, and baking quality were observed. However the biomass yield was 6% lower.     

Carbon partitioning to cellulose synthesis

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca²⁺ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis. Abbreviations: CesA, cellulose synthase; Csl, cellulose-like synthase (genes); DCB, dichlobenil; DPA, days after anthesis; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; P-SuSy, particulate SuSy; S-SuSy, soluble SuSy