Molecular evolution of the N-formyl peptide and C5a receptors in non-human primates

N-formyl peptides (FMLP) and complement fragment C5a are neutrophil chemoattractants. In humans, a single-copy gene was identified for the C5a receptor, and the receptor for FMLP (FPR1) is encoded by a single gene that shows 53% amino acid similarity to the C5aR. Two other humanFPR1 homologues,FPR-like 1 (FPR2/FPRL1) andFPR-like 2 (FPRL2) have been cloned. The human C5aR, FPR1, FPRL1, and FPRL2 are physically linked. By direct sequencing or by sequencing plasmid clones we studied theC5aR andFPR genes from four non-human primates (chimpanzee, gorilla, orangutan, and macaque). The sequences showed 95%–99% similarity to the human homologues, with the major divergences observed in macaque. In these genes, the transmembrane and the cytoplasmic domains are highly conserved, while the highest divergence corresponded to the extracellular loops involved in ligand binding. Additionally, we constructed a physical map of these genes in non-human primates. In all species the four genes were physically linked and we defined the relative orientation of the four genes in primates:C5aR>FPR1>FPR2 (FPRL1)>FPRL2. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have assigned the accession numbers X97730 (PTC5aR), X97731 (MMC5aR), X97732 (PPC5aR), X97730 (GGC5aR), X97734 (MMFPR1), X97735 (PPFPR1), X97736 (GGFPR1), X97737 (MMFPRL1), X97738 (GGFPRL1), X97739 (PTFPRL1), X97740 (MMFPRL2), X97741 (PPFPRL2), X97742 (GGFPRL2), X97743 (PTFPRL2), X97744 (PPFPRL1), and X97745 (PTFPR1)     

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the β-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized β-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation. Received: 22 December 1995 / Accepted: 12 March 1996     

The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, inSaccharomyces cerevisiae, theCDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence ofCDC40 (455 amino acids) contains four copies of a β-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in thecdc40-1 allele) results in normal vegetative growth at 23°C, and cell cycle arrest at 36°C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36°C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.     

Cloning and expression of the Neisseria meningitidis 5C outer membrane protein

The 5C outer membrane protein, one of the N. meningitidis class 5 proteins, was preferably expressed in bacteria isolated from the nasopharynx and its role in adhering to the mucosal cells and invading them as well as the development of anti-5C antibodies in healthy carriers was demonstrated. Anti-5C monoclonal antibodies are bactericidal in the presence of the human complement. The immunodominant region of the 5C protein is highly conserved among the different strains of N. meningitidis, and the opc gene, which encodes the protein, does not seem to show antigenic variations. Here the isolation of the opc gene from the Cuban strain B:4:P1.15 by PCR (Polymerase Chain Reaction) is presented. Under the regulation of the tryptophan promoter, the gene was cloned and sequenced in E. coli with a high level of expression and fused to the amino-terminal end of the interleukin-2 gene. In the dot-blot experiments, the presence of the gene in those strains which did not express the protein in the whole cell ELISA was also detectable.     

Phytoplankton strategies and time scales in a eutrophic reservoir

Phytoplankton species groups were studied in a eutrophic reservoir at different time scales (daily, weekly, monthly and yearly). Four strategic groups along the r-K continuum were defined and their seasonal time courses were followed. Their temporal distribution of relative biomass reflected resource partitioning because each strategic group dominated the phytoplankton community at different times in the year. However, the relationships between strategic groups changed with the time scale involved. At the daily scale an inverse relationship between r- and K-groups occurred whereas at supradaily scales such relationships did not hold. Species groups reflected strategic groups. No relationship between population growth and losses was found, suggesting that both were not counterbalanced. In the long term (supraannual) the phytoplankton community changed very much in this reservoir, its ecological memory being small as compared with that of lakes.     

Studies with Differentially Labeled [11C]Cocaine, [11C]Norcocaine, [11C]Benzoylecgonine,and [11C]-and 4′-[18F]Fluorococaine to Probe the Extent to Which [11C]Cocaine Metabolites Contribute to PET Images of the Baboon Brain

Abstract: The psychostimulant drug of abuse, cocaine (benzoylecgonine methyl ester), is rapidly metabolized by cleavage of its two ester groups, to give benzoylecgonine (BE) and ecgonine methyl ester, and by N-demethylation, to give N-norcocaine (NC). The recent use of [N-methyl-¹¹CH₃]cocaine to image brain cocaine binding sites with positron emission tomography (PET) raises the question of whether PET images partially reflect the distribution and kinetics of labeled cocaine metabolites. We prepared [O-metty/-¹¹CH₃]cocaine by methylation of the sodium salt of BE with [¹¹C]CH₃l, and showed that PET baboon brain scans, as well as regional brain kinetics and plasma time-activity curves corrected for the presence of labeled metabolites, are nearly identical to those seen with [N-methyl-¹¹CH₃]cocaine. This strongly suggests that ¹¹C metabolites do not significantly affect PET images, because the metabolite pattern is different for the two labeled forms of cocaine. In particular, nearly half the ¹¹C in blood plasma at 30 min was [¹¹C]CO₂ when [N-methy/-¹¹CH₃]cocaine was administered, whereas [¹¹C]CO₂ was not formed from [O-methy/-¹¹CH₃]cocaine. Only a trace of [¹¹C]NC was detected in plasma after [O-methyl-¹¹CH₃]cocaine administration. Nearly identical brain PET data were also obtained when 4′-[N-methy/-¹¹CH₃]fluorococaine and 4′-[¹⁸F]fluoro-cocaine (prepared by nucleophilic aromatic substitution from [¹⁸F]fluoride-and 4′-nitrococaine) were compared with [N-methy/-¹¹CH₃]cocaine. In vitro assays with rat brain membranes showed that cocaine and 4′-fluoroco-caine were equipotent at the dopamine reuptake site, but that 4′-fluorococaine was about 100 times more potent at the 5-hydroxytryptamine reuptake site. The studies with positron-emitting 4′-fluorococaines thus support the lack of significance of labeled metabolites or of binding to 5-hydroxytryptamine reuptake sites to PET images taken with [N-methy/-¹¹CH₃]cocaine. [¹¹C]NC prepared by O-methylation of norbenzoylecgonine gave PET images with preferential uptake in striatum, but slower clearance from all brain regions than [O-methy/-¹¹CH₃]cocaine. [¹¹C]BE prepared by N-methylation of norbenzoylecgonine did not show brain uptake.     

Improving cross-cultural skills of medical students through medical school-community partnerships.

Postulating that a program integrating language skills with other aspects of cultural knowledge could assist in developing medical students'' ability to work in cross-cultural situations and that partnership with targeted communities was key to developing an effective program, a medical school and two organizations with strong community ties joined forces to develop a Spanish Language and Hispanic Cultural Competence Project. Medical student participants in the program improved their language skills and knowledge of cultural issues, and a partnership with community organizations provided context and resources to supplement more traditional modes of medical education.     

Spatial, seasonal and long-term variability of phytoplankton photosynthesis in lakes

A cross-system, worldwide approach has been used to ascertainthe spatial, seasonal and long-term variability of areal phytoplanktonphotosynthesis (PP) in lakes using published data sets. Also,the average fraction of annual PP occurring under ice is calculated.The lakes considered embrace a range of properties (depth, mixing,flushing rate, latitude and trophic status). The overall yearlyPP distribution is skewed to the left, suggesting the dominanceof low PP rates in the data set. When comparing lake types,no differences in average PP have been found among them. Inparticular, there are no clear areal PP differences among lakesof different trophic status on yearly, averaged basis, suggestingthat environmental limitations to PP also exist in lakes ofhigher trophic status. Volumetric-based PP can be better usedto outline PP-based trophic differences, but some degree ofoverlap is also apparent. Across all lake types (except in tropicallakes), the PP seasonal course experiences only one peak inthe year, but its timing is clearly different for each laketype. The seasonal variability of PP is lower in tropical lakes,as previously reported, but the variability of the other laketypes is roughly the same. Therefore, the effects of depth,mixing regime, flushing rate and nutrient status on PP seasonalityare difficult to ascertain since they appear to be counterbalancedby other more pervasive, local effects. Particularly, thereis no increase in temporal variability with the trophic statusof lakes, suggesting that PP seasonal control by physical variablesoverrides that of nutrients. Also, no significant relationshipbetween average PP and latitude has been found. Seasonal variabilityincreases as the yearly PP increases. On a relative basis, thereis a spatial gradient of seasonal variability of PP, which isweaker when seasonal variability of PP is considered in interyearcomparisons. Long-term (i.e. interannual) variability of PPis clearly related to increasing yearly averaged PP. Specifically,in temperate, stratifying lakes the seasonal time course ofPP is clearly different from that of phytoplankton biomass,suggesting an uncoupling of both variables as a result of differingP_max and losses throughout the year. On an average basis, environmentalvariables are poor predictors of areal daily PP, thereby implyingthat the interplay of factors is complex and changing throughoutthe year. PP under ice averages 10% of yearly PP, but its variabilityis high enough to make its measurement advisable.     

Paracuaria hispanica n. sp. (Nematoda: Acuariidae), a stomach parasite of the pyrenean desman Galemys pyrenaicus Geoffr. (Insectivora: Talpidae), with a redefinition of the genus Paracuaria Rao, 1951

Adult and fourth-stage larvae of Paracuaria hispanica n. sp., from the stomach of the Pyrenean desman Galemys pyrenaicus Geoffroy (Insectivora: Talpidae) in northern and central Spain, are described. The new species differs from the other members of the genus Paracuaria (P. adunca and P. soricis), among other morphological details, in its smaller body and spicule sizes, the presence of a cuticular ring around the tip of the female tail, and the existence of lateral alae running longitudinally along its body from the cervical region to the tail. In view of the latter feature, the genus Paracuaria is redefined. The fourth stage larva of the new species is distinguished from that of P. adunca by its monocuspid deirids. P. hispanica occurred in 45% of the 20 host specimens examined.