The generic placement of a morphologically enigmatic species in Asteraceae: evidence from ITS sequences

 Bidens cordylocarpa is a high polyploid species restricted in distribution to stream sides in the mountains of Jalisco, Mexico. The morphologically enigmatic species was originally described as a member of the genus Coreopsis, but later transferred to Bidens, largely because the involucral bracts appear most similar to Bidens. Characters of the cypselae, often useful in generic placement, are of no value for this species because the fruits have features not detected in either Bidens or Coreopsis. Sequences from the internal transcribed spacer region of nuclear ribosomal DNA (ITS) were used to assess the relationships of Bidens cordylocarpa. The molecular phylogeny places B. cordylocarpa in a strongly supported clade of Mexican and South American Bidens, and provides more definitive evidence of relationships than morphology, chromosome number, or secondary chemistry. Molecular, morphological, and chromosomal data suggest that B. cordylocarpa is an ancient polyploid, perhaps the remnant of a polyploid complex. Received August 28, 2000 Accepted February 11, 2001     

Rapid, simple and sensitive high-performance liquid chromatographic method for detection and determination of acyclovir in human plasma and its use in bioavailability studies

A rapid, simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the measurement of acyclovir concentrations in human plasma and its use in bioavailability studies is evaluated. Unchanged acyclovir has been quantified without the introduction of an internal standard using the present method. Human plasma proteins were selectively precipitated by the addition of 7% perchloric acid to spiked plasma samples or to the plasma samples obtained after acyclovir administration to human volunteers and the mixture was spun at 1000 g for 10 min. The supernatant was directly injected into a Novaflex C₁₈ column and detected at 254 nm. The mobile phase consisted of octane sulfonic acid buffer (pH 2.5) and methanol (92:08). The limit of quantitation for acyclovir in plasma was 20 ng/ml, which enabled the determination of the area under the curve (AUC) more precisely, that is, it is much closer to its extrapolated value. The present method has been successfully applied to samples from bioavailability studies.     

A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA

A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary DNA oligomers to form specific cleavage and ligation substrates. It was, therefore, independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3' to 5' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and therefore the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on a agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in one tube in less than a day. The procedure was used to subclone and sequence the maize mitochondrial 18 S rDNA and 5' flanking region (2622 bases) in less than a week. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions, and cDNAs are discussed.     

High Altitude Frogs (Rana kukonoris) Adopt a Diversified Bethedging Strategy in the Face of Environmental Unpredictability

Environmental unpredictability can influence strategies of maternal investment among eggs within a clutch. Models predict that breeding females should adopt a diversified bet-hedging strategy in unpredictable environments, but empirical field evidence from Asia is scarce. Here we tested this hypothesis by exploring spatial patterns in egg size along an altitudinal gradient in a frog species(Rana kukunoris) inhabiting the Tibetan Plateau. Within-clutch variability in egg size increased as the environment became variable(e.g., lower mean monthly temperature and mean monthly rainfall at higher altitudes), and populations in environments with more unpredictable rainfall produced eggs that were smaller and more variable in size. We provide support for a diversified bet-hedging strategy in high-altitude environments, which experience dynamic weather patterns and therefore are of unpredictable environmental quality. This strategy may be an adaptive response to lower environmental quality and higher unpredictable environmental variance. Such a strategy should increase the likelihood of breeding success and maximize maternal lifetime fitness by producing offspring that are adapted to current environmental conditions. We speculate that in high-altitude environments prone to physical disturbance, breeding females are unable to consistently produce the optimal egg size due to physiological constraints imposed by environmental conditions(e.g., duration of the active season, food availability). Species and populations whose breeding strategies are adapted to cope with uncertain environmental conditions by adjusting offspring size and therefore quality show a remarkable degree of ability to cope with future climatic changes.     

Purification and characterization of carbon monoxide dehydrogenase, a nickel, zinc, iron-sulfur protein, from Rhodospirillum rubrum

Carbon monoxide dehydrogenase (CO dehydrogenase) from Rhodospirillum rubrum was shown to be an oxygen-sensitive, nickel, iron-sulfur, and zinc-containing protein that was induced by carbon monoxide (CO). The enzyme was purified 212-fold by heat treatment, ion-exchange, and hydroxylapatite chromatography and preparative gel electrophoresis. The purified protein, active as a monomer of Mr = 61,800, existed in two forms that were comprised of identical polypeptides and differed in metal content. Form 1 comprised 90% of the final activity, had a specific activity of 1,079 mumol CO oxidized per min-1 mg-1, and contained 7 iron, 6 sulfur, 0.6 nickel, and 0.4 zinc/monomer. Form 2 had a lower specific activity (694 mumol CO min-1 mg-1) and contained 9 iron, 8 sulfur, 1.4 nickel, and 0.8 zinc/monomer. Reduction of either form by CO or dithionite resulted in identical, rhombic ESR spectra with g-values of 2.042, 1.939, and 1.888. Form 2 exhibited a 2-fold higher integrated spin concentration, supporting the conclusion that it contained an additional reducible metal center(s). Cells grown in the presence of 63NiCl2 incorporated 63Ni into CO dehydrogenase. Although nickel was clearly present in the protein, it was not ESR-active under any conditions tested. R. rubrum CO dehydrogenase was antigenically distinct from the CO dehydrogenases from Methanosarcina barkeri and Clostridium thermoaceticum.     

Antibodies against calcium-regulating hormones in infectious-allergic forms of bronchial asthma

Antibodies to calcitonin, parathyroid hormone and cortisol are detected in acute stage of infection-caused bronchial asthma. The appearance of antibodies is paralleled by marked hypercalcaemia. The antibodies may bind excessive hormones in the blood, preventing further hormonal imbalance. Ten-day treatment with glucocorticoids decreased the amount of antibodies possibly due to normalization of hormonal secretion and restoration of their balance. As a result, calcium blood levels returned to normal.     

State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using saturation-transfer electron spin resonance

The state of aggregation of the (Ca2+ + Mg2+)-ATPase in the membrane of sarcoplasmic reticulum and in reconstituted membrane systems has been studied using saturation-transfer electron spin resonance (ST-ESR). Saturation-transfer ESR spectra show that in the sarcoplasmic reticulum, the ATPase is relatively free to rotate, with an effective rotational correlation time of approx. 33 microseconds at 4 degrees C, consistent with a monomeric or dimeric structure. The rate of rotation is observed to decrease with decreasing molar ratio of lipid to protein. In reconstituted systems, rotational motion of the ATPase on the millisecond time scale ceases when the lipids are in the gel phase. Addition of decavanadate, which causes the formation of crystalline arrays in negatively stained electron micrographs, results in only a small reduction in rotation rate for the ATPase in the membrane. The experiments are interpreted in terms of a short-lived (on the millisecond time scale) protein-protein interaction, with the formation of crystalline clusters of ATPase molecules which form and melt rapidly.     

Characterization of staphylococci

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.     

Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts

In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked.