Viability of basidiomycete strains after cryopreservation: comparison of two different freezing protocols

The viability of 250 basidiomycete strains was determined after a 2-d and then after a 2-year storage under liquid nitrogen using two different freezing protocols. Using an original agar plug protocol (OP), 162 strains (65%) of the 250 strains survived a 2-d storage and 158 strains (63%) survived a 2-year storage in liquid nitrogen. Using a straw protocol (CP), 246 strains (98%) of the 250 strains survived a 2-d storage and 243 strains (97%) a 2-year storage in liquid nitrogen. In addition, other 106 strains were newly estimated using the CP protocol; 104 (98%) of them survived successfully a 2-d storage and 101 (95%) of them survived a 2-year storage in liquid nitrogen. The results indicate that the protocol used for cryopreservation can significantly influence strain survival. Markedly better results were obtained using the CP protocol.     

Chromosome study of Lithobius forficatus (Lithobiomorpha, Chilopoda)

The diploid number 2n = 46 and the chromosome arm number NF = 74 are described in Lithobius forficatus from Olsztyn (Poland). Analyses of silver and CMA3-stained mitotic chromosomes suggest that a single chromosome pair has active NORs which correspond to G-C-rich (CMA3-positive) chromatin. Heteromorphism of the largest metacentric chromosome pair was observed. The sex chromosomes were not identified. Size polymorphism of the first chromosome pair was found.     

Synthesis and X-ray structures of sulfate esters of fructose and its isopropylidene derivatives. Part 1: 2,3:4,5-di-O-isopropylidene-beta-D-fructopyranose 1-sulfate and 4,5-O-isopropylidene-beta-D-fructopyranose 1-sulfate

2,3:4,5-Di-O-isopropylidene-beta-D-fructopyranose 1-sulfate have been synthesized by treatment of 2,3:4,5-di-O-isopropylidene-beta-D-fructopyranose with pyridine-sulfur trioxide complex. Direct hydrolysis of the isopropylidene group at C-4, C-5 gave 2,3-O-isopropylidene-beta-D-fructopyranose 1-sulfate. The crystal and molecular structures of ammonium (1a) and potassium (1b) salts of diisopropylidene derivative and ammonium (2) salt of monoisopropylidene derivative were determined by X-ray crystallography. Data for 1a and 1b were collected in 120 K and in 150 K for 2. All salts crystallized in P2(1)2(1)2(1) space group. There are three independent anions in asymmetric unit in 1b. Pyranose rings in the diisopropylidene derivative salts studied adopt 2S(0) twist boat conformation, whereas in the monoisopropylidene exists in a slightly distorted chair conformation (4C(1)). A staggered conformation is preferred by the sulfate group as indicated by values of C-(ester)-S-O(terminal) torsion angles: -173.2(4) degrees in 1a, 175.1(6) degrees in anion A of 1b, 170.8(6) degrees in anion C of 1b and 177.9(2) degrees in 2. However, strong interactions such as potassium-oxygen and H-bonds may affect the geometry: in anion B of 1b the value of the torsion angle is 139.4(6) degrees.     

Evolutionary dynamics and population control during in vitro selection and amplification with multiple targets

Iterative cycles of in vitro selection and amplification allow rare functional nucleic acid molecules, aptamers, to be isolated from large sequence pools. Here we present an analysis of the progression of a selection experiment that simultaneously yielded two families of RNA aptamers against two disparate targets: the intended target protein (B52/SRp55) and the partitioning matrix. We tracked the sequence abundance and binding activity to reveal the enrichment of the aptamers through successive generations of selected pools. The two aptamer families showed distinct trajectories of evolution, as did members within a single family. We also developed a method to control the relative abundance of an aptamer family in selected pools. This method, involving specific ribonuclease digestion, can be used to reduce the background selection for aptamers that bind the matrix. Additionally, it can be used to isolate a full spectrum of aptamers in a sequential and exhaustive manner for all the different targets in a mixture.     

Glucocorticoid regulation and glycosylation of mouse intestinal type IIb Na-P(i) cotransporter during ontogeny

We sought to characterize expression of an apically expressed intestinal Na-P(i) cotransporter (Na-P(i)-IIb) during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na-P(i) uptake by intestinal brush-border membrane vesicles was highest at 14 days of age, lower at 21 days, and further reduced at 8 wk and 8-9 mo of age. Na-P(i)-IIb mRNA and immunoreactive protein levels in 14-day-old animals were markedly higher than in older groups. MP treatment significantly decreased Na-P(i) uptake and Na-P(i)-IIb mRNA and protein expression in 14-day-old mice. Additionally, the size of the protein was smaller in 14-day-old mice. Deglycosylation of protein from 14-day-old and 8-wk-old animals with peptide N-glycosidase reduced the molecular weight to the predicted size. We conclude that intestinal Na-P(i) uptake and Na-P(i)-IIb expression are highest at 14 days and decrease with age. Furthermore, MP treatment reduced intestinal Na-P(i) uptake approximately threefold in 14-day-old mice and this reduction correlates with reduced Na-P(i)-IIb mRNA and protein expression. We also demonstrate that Na-P(i)-IIb is an N-linked glycoprotein and that glycosylation is age dependent.     

Analysis of the catalytic domain of phosphatidylinositol 4-kinase type II

Phosphatidylinositol (PtdIns) 4-kinases catalyze the conversion of PtdIns to PtdIns 4-phosphate, the major precursor of phosphoinositides that regulates a vast array of cellular processes. Based on enzymatic differences, two classes of PtdIns 4-kinase have been distinguished termed Types II and III. Type III kinases, which belong to the phosphatidylinositol (PI) 3/4-kinase family, have been extensively characterized. In contrast, little is known about the Type II enzymes (PI4KIIs), which have been cloned and sequenced very recently. PI4KIIs bear essentially no sequence similarity to other protein or lipid kinases; hence, they represent a novel and distinct branch of the kinase superfamily. Here we define the minimal catalytic domain of a rat PI4KII isoform, PI4KIIalpha, and identify conserved amino acid residues required for catalysis. We further show that the catalytic domain by itself determines targeting of the kinase to membrane rafts. To verify that the PI4KII family extends beyond mammalian sources, we expressed and characterized Drosophila PI4KII and its catalytic domain. Depletion of PI4KII from Drosophila cells resulted in a severe reduction of PtdIns 4-kinase activity, suggesting the in vivo importance of this enzyme.     

Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.     

Genome-wide patterns of expression in Drosophila pure species and hybrid males

One of the most fundamental questions for understanding the origin of species is why genes that function to cause fertility in a pure-species genetic background fail to produce fertility in a hybrid genetic background. A related question is why the sex that is most often sterile or inviable in hybrids is the heterogametic (usually male) sex. In this survey, we have examined the extent and nature of differences in gene expression between fertile adult males of two Drosophila species and sterile hybrid males produced from crosses between these species. Using oligonucleotide microarrays and real-time quantitative polymerase chain reaction, we have identified and confirmed that differences in gene expression exist between pure species and hybrid males, and many of these differences are quantitative rather than qualitative. Furthermore, genes that are expressed primarily or exclusively in males, including several involved in spermatogenesis, are disproportionately misexpressed in hybrids, suggesting a possible genetic cause for their sterility.     

Subcellular targets of mercaptoborate (BSH), a carrier of 10B for neutron capture therapy (BNCT) of brain tumors

The transformed C6 glial cells in cultures were treated with sodium mercaptoborate (Na(2)B(12)H(11)SH, BSH), a carrier of atomic targets ((10)B) of thermal neutrons for the neutron capture therapy of brain tumors. As shown by light microscopy, the therapeutic dose of BSH (100 microg/ml) did not alter the gross morphology and growth of the population of cells within a 72 h treatment interval. Electron microscopic analysis of these cells revealed activation of nucleoli and, occasionally, enlarged and bifurcated mitochondria. After 200 microg BSH/ml and 72 h treatment, growth of the cell population was inhibited and ultrastructural changes became more profound. They included condensation of chromatin and its allocation to the nuclear envelope which formed deeper invaginations. Mitochondria further increased in size and were characterized by slim or angular cristae. Moreover, in circumscribed segments of some of the slightly swollen mitochondria their cristae disappeared or were reduced to fine pouch-like structures localized near the continuous outer membrane, suggestive for a non-destructive restructuring of the inner mitochondrial membrane. The smooth pinocytotic vesicles near the plasma membrane, lysosomes and heterogeneous dense bodies were more frequent. The revealed subcellular targets of BSH may initiate the development of pharmacological protocols aimed to further improve the tolerance to BSH by the healthy tissues of patients undergoing BNCT of brain tumors, e.g. by intervention into the oxidative stress triggered likely by the altered mitochondria.