Complex segregation analysis of non-myoclonic idiopathic generalized epilepsy in families ascertained from probands affected with idiopathic epilepsy with tonic-clonic seizures in Antioquia,Colombia

In an attempt to identify the possible role of major genes, multifactorial inheritance, and cohort effects in the susceptibility to idiopathic epilepsy with generalized tonic-clonic seizures of the awakening type (GTCS), complex segregation analysis was performed in 196 nuclear families ascertained through affected probands with idiopathic epilepsy with GTCS belonging to the Paisa community of Antioquia (Colombia). Models postulating no transmission, single major locus (dominant and recessive) only, and multifactorial component only, were rejected. Since the codominant single major locus model could not be rejected and models that assign no major locus to transmission, no polygenic component to transmission, and no transmission of the major effect were rejected, complex segregation analysis suggested that a major autosomal codominant allele together with a multifactorial component (mixed model) best explained clustering of idiopathic epilepsy with GTCS in families of the Paisa community. The deficit of transmission of heterozygotes (0.17) is compatible with the existence of epistasis acting on a major gene whose frequency was estimated to be 0.0211. Its transmission variance accounts for 81% of the susceptibility to idiopathic epilepsy with GTCS. The complementary variance (19%) is due to the polygenic component. Received: 19 January 1996 / Revised: 11 March 1996     

Temporal Variation of Allozyme Frequencies in Aedes albifasciatus (Diptera: Culicidae) from Argentina

Aedes albifasciatus is a floodwater mosquito widely distributed in Argentina. It is important from economic and medical points of view. A 4-year survey of seasonal variation in allele frequencies in a population of this species was undertaken to determine possible changes in the genetic structure and their correlation with environmental conditions. Significant temporal variation was detected at most of the loci, but it did not follow a cyclic or seasonal pattern. Multivariate analysis of principal components showed a remarkable homogeneity of samples collected from December 1993 to April 1995 and a clear differentiation of the November 1991, March 1992, and November 1993 samples. This variation could be correlated with the magnitude of rainfall occurring in the area. Passive transport of larvae by water streams and river freshets produced by floods would have mixed larvae from breeding sites with different allele frequencies, causing the genetic differentiation observed.     

Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog.

We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borrelia hermsii. The 40-kDa protein was solubilized from whole organisms with 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chromosomal DNA lambda EXlox expression library and identified by using affinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide encoding a putative leader peptidase II cleavage site, indicating that the 40-kDa protein was a lipoprotein. Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosphodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), and Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein a glycerophosphodiester phosphodiesterase (Gpd) homolog, the first B. hermsii lipoprotein to have a putative functional assignment. A nonlipidated form of the Gpd homolog was overproduced as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to immunize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and conversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constituent of purified outer membrane vesicles prepared from B. hermsii. Screening of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, Treponema pallidum, and Leptospira kirschneri. Further sequence analysis both upstream and downstream of the Gpd homolog showed additional homologs of glycerol metabolism, including a glycerol-3-phosphate transporter (GlpT), a glycerol-3-phosphate dehydrogenase (GlpD), and a thioredoxin reductase (TrxB).     

Purification and properties of xylanase A from alkali-tolerant Bacillus sp. strain BP-23.

Xylanase A from the recently isolated Bacillus sp. strain BP-23 was purified to homogeneity. The enzyme shows a molecular mass of 32 kDa and an isoelectric point of 9.3. Optimum temperature and pH for xylanase activity were 50 degrees C and 5.5 respectively. Xylanase A was completely inhibited by N-bromosuccinimide. The main products of birchwood xylan hydrolysis were xylotetraose and xylobiose. The enzyme was shown to facilitate chemical bleaching of pulp, generating savings of 38% in terms of chlorine dioxide consumption. The amino-terminal sequence of xylanase A has a conserved sequence of five amino acids found in xylanases from family F.     

Ultraviolet-visible absorption spectrum for the complex phosphorylase b—AMP: study at 25°C

Absorption difference spectra of phosphorylase b when AMP binds to its high affinity site have been studied at 25°C and pH 6.9; the absorbance changes show linearity as a function of the amount of phophorylase b—AMP complex present in solution. The negative regions of these spectra have been interpreted by assigning the hypochromic effect in the absorption band of AMP to stacking of the adenine ring with an aromatic ring from some tyrosine and/or tryptophan residues. On the other hand, the positive region of the difference spectrum induced by binding of AMP to its high affinity site can be simulated assuming that six tyrosines and one or two tryptophans per monomer are embedded in a highly hydrophobic environment.     

Quantitative aspects of triacylglycerol metabolism and sterol synthesis from 14C-acetate in etiolated seedlings of Euphorbia lambii

Triacylglycerols occur in both the endosperm and embryo of Euphorbia lambii seeds. Upon germination, the amount of these neutral lipids in the endosperm decreased with 1.06 mg fatty acid day^-1. The embryo contained 1.4 mg fatty acids in the triacylglycerols and this value declined slowly to 0.4 mg seedling^-1 during the 8 day period of endosperm depletion. Radioactive acetate was rapidly taken up by the cotyledons of intact seedlings, translocated throughout the entire seedling, and up to 10.5% of the ¹⁴C proceeded to the sterols and latex triterpenols. Maximum uptake values of 1.4 μmol seedling^-1 day^-1 of acetate were measured. Acetate uptake and subsequent incorporation into sterols and triterpenols decreased substantially in the presence of increasing amounts of sucrose (up to 0.3 M). Traces of acetate did not effect [¹⁴C]-sucrose uptake and corresponding synthesis of [¹⁴C]-sterols and triterpenols, but increased concentrations of acetate (0.05 M and up) reduced both uptake of sucrose and its conversion into unsaponifiable lipids.
The uptake capacity of the cotyledons for [¹⁴C]-glycerol exceeded the daily production in the endosperm, but only a small amount of label proceeded to the sterols and triterpenols. [¹⁴C]-Triacylglycerols were never detected in the seedling, regardless of the labeled substrate used. Although acetate is an efficient precursor in triterpenol and sterol synthesis, the uptake capacity of the cotyledons for this metabolite is too small in relation to the daily production of water soluble substrates in the endosperm. If acetate is released by the endosperm, only a marginal contribution towards triterpenol and sterol synthesis in the seedling is to be anticipated from this substrate.     

Characterization of an endoserine protease secreted by Arthrobacter aureus.

A 22-kDa endoserine protease secreted by an Arthrobacter aureus strain was identified. The optimal temperature for this protease was 70 degrees C. Protease production was maximal at the end of the exponential growth phase, and the protease was induced by amino acids and peptides and repressed by carbon sources.     

Application of paclobutrazol and GA3 to adult peach trees: Effects on nutritional status and photosynthetic pigments

Soil applications of 1 or 2 g paclobutrazol (Pbz) and foliar sprays of 1000 mg L^-1 GA₃ to adult peach trees [Prunus persica (L.) Batsch] had opposite effects: vegetative growth was inhibited by paclobutrazol and promoted by GA₃, whereas mean fruit yield was greater in Pbz-treated trees and less in GA₃-sprayed trees. Compared to controls, differences in the concentration of different mineral nutrients occurred in leaves collected shortly after fruit harvest: in Pbz-treated trees, the concentration of N and K significantly decreased, whereas that of Mg and Mn increased. Leaves of trees sprayed with GA₃ had a significantly lower concentration of N and Ca and Mn, and a slightly greater concentration of K. In spite of these changes, element concentrations were within accepted ranges and therefore the nutritional status of peach trees did not change with the application of both growth regulators. Pbz did not alter the concentrations of chlorophylls, whereas GA₃ significantly reduced the concentrations of chlorophylls a and b. Neoxanthin, violaxanthin, antheraxanthin, lutein, zeaxanthin, and ß-carotene concentrations were unaffected by all treatments.Mention of trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the C.S.I.C. (Ministry of Education, Spain) and does not imply its approval to the exclusion of other products or vendors that may be suitable.     

Synthesis of ribosomal proteins during growth of Streptomyces coelicolor

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [³⁵ S]-methionine, separated by two-dimensional poly-acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ-galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.     

Over-expression of human type I (placental) 3β-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus

Human type I placental 3β-hydroxy-5-ene-steroid dehydrogenase/steroid 5→4-ene-isomerase (3β-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3β-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3β-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3β-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3β-HSD substrate, 5-androstan-3β-o1-17-one, in the Sf-9 cell homogenate (K_m = 17.9 μM) and placental microsomes (K_m = 16.7 μM). The 3β-HSD activity (V_max = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (V_max = 9.1 nmol/min/mg). The K_m values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (K_m = 14.7 μM) and placental microsomes (K_m = 14.4 μM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (V_max = 25.7 nmol/min/mg) relative to placenta (V_max = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3β-HSD/isomerase in human placenta.