Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes

Oocytes induced to undergo meiotic maturation by progesterone possess a cytoplasmic activity that causes germinal vesicle breakdown (GVBD). The cytoplasmic factor postulated to be responsible for this activity is designated as the maturation promoting factor (MPF). The activity of MPF was assayed by injecting cytoplasm into fully-grown oocytes to induce GVBD. It was found that maturing oocyte cytoplasm possesses MPF activity before GVBD begins. Treatment of progesterone stimulated oocytes with cycloheximide, either applied externally or injected, inhibited the appearance of MPF in the cytoplasm as well as GVBD when the inhibitor treatment was initiated before the cytoplasm exhibited MPF activity. In contrast, the same treatment did not inhibit GVBD when it was applied to oocytes after the cytoplasm possessed MPF activity. Furthermore, cycloheximide treatment of recipient oocytes did not inhibit the induction of GVBD by injected cytoplasm containing MPF. Cytoplasm of oocytes injected with MPF subsequently possessed MPF activity as high as that of the original donor cytoplasm in spite of its extensive dilution. This suggests that amplification of MPF took place in the recipient. Cycloheximide treatment did not inhibit the amplification of MPF. It was concluded that cycloheximide inhibits only the initial phase of induction of MPF activity, but neither its amplification nor its action on the nucleus that causes GVBD. From these results, a hypothesis concerning the cytoplasmic mechanism for the induction of GVBD has been proposed.     

Fourier transform infrared study of photoreceptor membrane. I. Group assignments based on rhodopsin delipidation and reconstitution

Fourier transform infrared spectroscopy has been used to study the structure of bovine photoreceptor membrane. Rhodopsin appears to contain an extensive alpha-helical structure which is arranged predominantly perpendicular to the membrane plane. Spectra of delipidated rhodopsin and rhodopsin membranes reconstituted from dioleyl-phosphatidylcholine were compared with native photoreceptor membrane from rod outer segments in order to facilitate peak assignments. It is concluded that spectroscopic peaks characteristic of several protein and lipid groups can be assigned. We also find delipidation leads to alteration of the rhodopsin structure which is restored upon reconstitution. Membranes both suspended in ²H₂O and dehydrated were compared in order to detect possible conformational differences. Dehydration does not appear to grossly alter rhodopsin structure, although it may affect delipidated rhodopsin.     

Phylogenetic relationships within the aplocheiloid fish genus Rivulus (Cyprinodontiformes, Rivulidae): implications for Caribbean and Central American biogeography

We examined the phylogenetic relationships of 16 northern species of the aplocheiloid genus Rivulus inhabiting the Caribbean, Central America, and South America. A total of 714 base pairs per taxon were sequenced from two segments of the mitochondrial genome, 12S rRNA and cytochrome b. Both parsimony and neighbor-joining analyses suggest an ancient vicariant origin of the Greater Antillean taxa, in addition to a quite recent dispersal of species into the Lesser Antilles from the South American mainland. Combined analyses support the monophyly of the northern South American assemblage as the sister group of a Central American/Columbian biota. However, the monophyly of the Central American biota remains uncertain. Divergence estimates for the Central American taxa are calibrated from the Last Cretaceous separation of the proto-Antilles from the Americas. These data suggest that the extant Central American taxa represent the descendants of at least two separate invasions during the Cenozoic, prior to the closing of the Panamanian isthmus. Times are consistent with the extensive evidence for reptilian and mammalian exchange throughout the Cenozoic.      

Modeling residue usage in aligned protein sequences via maximum likelihood

A computational method is presented for characterizing residue usage, i.e., site-specific residue frequencies, in aligned protein sequences. The method obtains frequency estimates that maximize the likelihood of the sequences in a simple model for sequence evolution, given a tree or a set of candidate trees computed by other methods. These maximum- likelihood frequencies constitute a profile of the sequences, and thus the method offers a rigorous alternative to sequence weighting for constructing such a profile. The ability of this method to discard misleading phylogenetic effects allows the biochemical propensities of different positions in a sequence to be more clearly observed and interpreted.      

Photoactivation of rhodopsin causes an increased hydrogen-deuterium exchange of buried peptide groups.

A key step in visual transduction is the light-induced conformational changes of rhodopsin that lead to binding and activation of the G-protein transducin. In order to explore the nature of these conformational changes, time-resolved Fourier transform infrared spectroscopy was used to measure the kinetics of hydrogen/deuterium exchange in rhodopsin upon photoexcitation. The extent of hydrogen/deuterium exchange of backbone peptide groups can be monitored by measuring the integrated intensity of the amide II and amide II' bands. When rhodopsin films are exposed to D2O in the dark for long periods, the amide II band retains at least 60% of its integrated intensity, reflecting a core of backbone peptide groups that are resistant to H/D exchange. Upon photoactivation, rhodopsin in the presence of D2O exhibits a new phase of H/D exchange which at 10 degrees C consists of fast (time constant approximately 30 min) and slow (approximately 11 h) components. These results indicate that photoactivation causes buried portions of the rhodopsin backbone structure to become more accessible.     

Late thrombotic occlusion of a malapposed stent 10 months after intracoronary brachytherapy

We report a patient who received a stent following intracoronary 3-irradiation. Despite a good initial angiographic result, the stent appeared to be not fully expanded on intravascular ultrasound imaging at 6-month follow-up. Four months later, sudden thrombotic occlusion occurred shortly after aspirin cessation.     

Species-specific sperm adhesion in sea urchins. A quantitative investigation of bindin- mediated egg agglutination

Bindin, a protein component of the acrosomal vesicle of sea urchin sperm, has been isolated from Arbacia punctulata and strongylocentrous purpuratus. Using this isolated bindin, we have devised a quantitative assay for bindin-mediated egg agglutination and compared the agglutination of bindin eggs from A. puntulata and S. purpuratus. Bindin- mediated agglutination is species –specific in both species, although a measurable degree of heterotypic interaction is observed. Homotypic bindin-egg interactions differ significantly from heterotypic interactions both in the extent of agglutination and the size of the resulting aggregates. We also provide direct evidence that bindin particles agglutinate eggs by adhering to the surfaces of adjacent eggs. Although the A. punctulata bindin preparation displays the same functional properties and consists of one major polypeptide of the same apparent molecular weight as S. purpuratus bindin, its morphology is very different. Unlike the spherical aggregates observed with S. purpuratus bindin, A punctulata bindin exists as lamellar vesicles and binds significant amounts of phospholipids and Triton X-100, suggesting that it may be tightly associated with the acrosomal membrane. Having defined a number of the basic parameters of bindin-mediated agglutination, we examined the effect of a number of saccharides and glycopeptides on bindin-mediated egg agglutination. Carbohydrate-containing components derived from the egg cell surface by proteolysis were found to inhibit bindin-mediated egg agglutination at low concentrations, but this inhibition is not species specific.     

Monitoring of biomass composition from microbiological sources by means of FT‐IR spectroscopy

An FT‐IR spectroscopic method was developed for the simultaneous quantitative analysis of biomacromolecular components in biomass, originating from various microbiological sources. For the determination of protein, lipid and carbohydrate content, creatine phosphokinase, egg phosphatidyl choline and starch hydrolysate were chosen as external standards. This selection was based on spectral similarity and ease of availability. Protein content was based on the area under the amide II band profile around 1,545 cm^?1. Because of the heterogeneous lipid composition in the different species, lipid content was determined using integration over the C? H stretching vibrational population between 2,984 and 2,780 cm^?1. Carbohydrate content was determined using integration over a C? O and C? O? C stretching band area between 1,180 and 1,133 cm^?1. Linear regression analysis provided three calibration lines, according to which biomasses from ten species were analyzed. This approach showed good intra‐batch reproducibility. With this method we could demonstrate good reproducibility between batches of the same species with similar growth conditions while large differences in biomass composition were observed between the various species. Protein content as determined by FT‐IR spectroscopy compared well with the results obtained from elemental analysis. Biotechnol. Bioeng. 2009;103: 123–129. © 2008 Wiley Periodicals, Inc.     

An intragenic distribution bias of DNA uptake sequences in <Emphasis Type="Italic">Pasteurellaceae</Emphasis> and <Emphasis Type="Italic">Neisseriae</Emphasis>

   

Most sequenced strains from Pasteurellaceae and Neisseriae contain hundreds to thousands of uptake sequence (US) motifs in their genome, which are associated with natural competence for DNA uptake. The mechanism of their recognition is still unclear, and I searched for intragenic location patterns of these motifs for clues about their distribution. In all cases, one orientation of the US has a higher occurrence in the reading frame, and in all Pasteurellaceae, the US and the reverse complement motifs are biased towards the gene termini. These findings could help design experimental set-ups to study preferential DNA uptake, thereby further unravelling the phenomenon of natural competence.