The reported point centromeres of Scheffersomyces stipitis are retrotransposon long terminal repeats

Point centromeres, found in some ascomycete yeasts such Saccharomyces cerevisiae, are very different in structure from the centromeres of other eukaryotes. They are tiny and nonrepetitive and contain only two short conserved sequence motifs. Until recently, point centromeres were thought to have a single evolutionary origin, in the budding yeast family Saccharomycetaceae. Most yeasts outside this family have centromeres that are many kilobases in size. Some have centromeres consisting of a large inverted repeat sequence, others have centromeric clusters of retrotransposons, and a third group including Candida albicans has centromeres with no conserved sequence features. It was recently reported that Scheffersomyces stipitis has point centromeres with a strongly conserved 125-bp core sequence, which is unexpected because S. stipitis is only distantly related to the known point-centromere species. We show here that the 125-bp core sequence is actually part of the long terminal repeat (LTR) of the Ty5-like retrotransposon Tps5, which forms a cluster in the centromeric region of each S. stipitis chromosome. Thus, the LTR of a centromere-associated retrotransposon confers centromere-like mitotic stability when cloned into a plasmid. The centromeric regions of S. stipitis contain three types of Tps5 element (Tps5a, Tps5b, and Tps5c) and a noncoding nonautonomous large retrotransposon derivative.     

Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase

Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to assemble as a stable complex on the donor substrate and to catalyze strand transfer. We now demonstrate that subsequent to assembly, catalysis of both 3' end processing and strand transfer requires a divalent cation cofactor and that the divalent cation requirements for assembly and catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cation requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end processing and strand transfer assays are then used in conjunction with exonuclease III protection analysis to investigate the effects of integrase inhibitors on each step in the reaction. Analysis of a series of related inhibitors demonstrates that these types of compounds affect assembly and not either catalytic process, therefore reconciling the apparent disparate results obtained for such inhibitors in assays using isolated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysis and have implications for both the identification and characterization of integrase inhibitors.     

Effects of vasodilators and perfusion pressure on coronary flow and simultaneous release of nitric oxide from guinea pig isolated hearts

OBJECTIVE: The aims were to validate the use of a direct reading NO electrode, to compare the effects of diverse acting drugs on altering coronary flow (CF) and NO release, and to examine the effects of altered perfusion pressure on flow-induced changes in NO concentration [NO] in the hemoglobin free effluent of guinea pig isolated hearts. METHODS: Hearts were isolated and perfused initially at a constant perfusion pressure (55 mmHg) with a modified Krebs-Ringer's solution equilibrated with 97% O2 and 3% CO2 at 37 degrees C. Heart rate, left ventricular pressure, CF, and effluent pH, pCO2, pO2, and NO generated current were monitored continuously on-line. Effluent was sampled for L-citrulline. Percent O2 extraction and O2 consumption were calculated. [NO] was quantitated with a sensitive amperometric sensor (sensitivity > or = 1 nmol/l approximately 3 pA) and a selective gas permeable membrane. RESULTS: The electrode was not sensitive to changes in solution pO2, flow, or pressure. The electrode was sensitive to pCO2 (-0.50 nmol/l/mmHg) and temperature (+24.5 nmol/l/degree C), so coronary effluent pCO2 was measured to compensate for a small decrease in pCO2 that occurred with an increase in coronary flow, and effluent temperature was rigidly controlled. Serotonin, bradykinin, and nitroprusside increased NO release along with CF, whereas nifedipine, butanedione monoxime, zaprinast, and bimakalim comparably increased CF but did not increase [NO] or NO release. Increases in CF (ml/g/min) and NO release (pmol/g/min), respectively, were 5.0 +/- 1 and 100 +/- 17 for 1 mumol/l serotonin, 7.5 +/- 1 and 148 +/- 18 for 100 nmol/l bradykinin, and 7.8 +/- 1 and 173 +/- 28 for 100 mumol/l nitroprusside. The increases in effluent NO by bradykinin were proportional to the increases in L-citrulline. Tetraethylammonium decreased CF, but did not change NO release, indomethacin changed neither CF nor NO release, and NG-nitro-L-arginine methyl ester (L-NAME) reduced CF by 2.6 +/- 1 ml/g/min and NO release by 25 +/- 8 pmol/g/min. An increase of CF of 8.0 +/- 0.3 ml/g/min, produced by increasing perfusion pressure from 25 to 90 mmHg, increased [NO] by 30 +/- 4 nmol/l; L-NAME but did not reduce the pressure-induced increase in CF, but reduced the increase in [NO] to 10 +/- 5 nmol/l. CONCLUSIONS: This study demonstrates in intact hearts real-time release of NO by several vasodilator drugs and by pressure-induced increases in flow (shear stress) and attenuation of these effects by L-NAME.     

K-winner networks.

A special class of mutually inhibitory networks is analyzed, and parameters for reliable K-winner performance are presented. The network dynamics are modeled using interactive activation, and results are compared with the sigmoid model. For equal external inputs, network parameters that select the units with the larger initial activations (the network converges to the nearest stable state) are derived. Conversely, for equal initial activations, networks that select the units with larger external inputs (the network converges to the lowest energy stable state) are derived. When initial activations are mixed with external inputs, anomalous behavior results. These discrepancies are analyzed with several examples. Restrictions on initial states are derived which ensure accurate K-winner performance when unequal external inputs are used.     

A role for Edman degradation in proteome studies

Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two-dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottleneck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, an N-terminal sequence tag.     

Analysis of immunoglobulin-synthesizing cells in human dental periapical lesions by in situ hybridization and immunohistochemistry

The iron status of 22 children and adolescents with Crohn's disease (mean age: 13 years) was evaluated. Eleven patients were suffering from active disease with inflammation, identified by at least one abnormal value for serum orosomucoid, C-reactive protein or sedimentation rate (group I). Eleven patients were in clinical remission and showed no biological evidence of inflammation (group II). Hemoglobin and red cell indices, erythrocyte protoporphyrin, serum iron, transferrin, serum ferritin and basic red cell ferritin were determined in all patients. The usual indicators of iron status, particularly serum ferritin, were affected by the inflammatory processes, but basic red cell ferritin appeared to be independent of inflammation. Basic red cell ferritin can therefore be considered to be a reliable indicator of iron status in children and adolescents with Crohn's disease.