A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants. In this paper, we derive a new PtdIns4P biosensor using the PtdIns4P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila. PtdIns4P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7-positive late endosomes/lysosomes. PtdIns4P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. Therefore, P4M reports a wider cellular distribution of PtdIns4P than previous probes and therefore will be valuable for dissecting the biological functions of PtdIns4P in its assorted membrane compartments.     

RNA–Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel

Knowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (¹³C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [¹³C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect ¹³C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed that Bacteroides uniformis, Blautia glucerasea, Clostridium indolis, and Bifidobacterium animalis were the main users of the ¹³C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified. B. uniformis utilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.     

Curiously Modern DNA for a ``250 Million-Year-Old' Bacterium

Studies of ancient DNA have attracted considerable attention in scientific journals and the popular press. Several of the more extreme claims for ancient DNA have been questioned on biochemical grounds (i.e., DNA surviving longer than expected) and evolutionary grounds (i.e., nucleotide substitution patterns not matching theoretical expectations for ancient DNA). A recent letter to Nature from Vreeland et al. (2000), however, tops all others with respect to age and condition of the specimen. These researchers extracted and cultured a bacterium from an inclusion body from what they claim is a 250 million-year (Myr)-old salt crystal. If substantiated, this observation could fundamentally alter views about bacterial physiology, ecology and evolution. Here we report on molecular evolutionary analyses of the 16S rDNA from this specimen. We find that 2-9-3 differs from a modern halophile, Salibacillus marismortui, by just 3 unambiguous bp in 16S rDNA, versus the ∼59 bp that would be expected if these bacteria evolved at the same rate as other bacteria. We show, using a Poisson distribution, that unless it can be shown that S. marismortui evolves 5 to 10 times more slowly than other bacteria for which 16S rDNA substitution rates have been established, Vreeland et al.'s claim would be rejected at the 0.05 level. Also, a molecular clock test and a relative rates test fail to substantiate Vreeland et al.'s claim that strain 2-9-3 is a 250-Myr-old bacterium. The report of Vreeland et al. thus falls into a long series of suspect ancient DNA studies. Received: 12 April 2001 / Accepted: 9 June 2001     

The effects of estrogen on cortisol metabolism in female baboons

We examined Cortisol (F) dynamics in female baboons $\text{(Papio anubis)}$ treated with diethylstilbestrol (DES) or estradiol (E₂) and compared values with those previously measured in nonpregnant and pregnant animals. Five regularly menstruating baboons (12–18 kg, BW) were administered 5 mg DES daily via fruit or 0.5 mg E₂/0.1ml oil sc for 30 days. Blood samples, obtained before and after treatment, were assayed for serum F concentrations and serum Cortisol binding capacity (CBC). The metabolic clearance (MCR) and production rate (PR) of F and the catabolism of i.v. administered [³H] F were examined 25 and 30 days after initiation of estrogen treatment. Compared with values in nonpregnant baboons, F metabolism in estrogen treated animals is significantly altered and is characterized by increased formation of unconjugated metabolites, decreased glucuronylation, increased excretion of unconjugated F, cortisone, and highly polar metabolites, and increased CBC. These changes induced by estrogen are similar to those observed in intact pregnant baboons and permit the suggestion that the pattern of F metabolism and the level of CBC in baboon pregnancy are the result of elevated estrogen production.However, estrogen also caused a significant decrease in the MCR and PR of F, parameters which, by contrast, are similar in intact pregnant and nonpregnant baboons. These findings indicate that while estrogen also influences the rate of F clearance and F production, these effects of estrogen are not apparent during pregnancy. Collectively, these findings allow the suggestion that estrogen is a major factor which alters F metabolism and increases serum CBC in baboon gestation. However, additional factors are operative in primate pregnancy which maintain PR and MCR of F at levels similar to those of nonpregnant baboons.     

Gene N regulator function of phage λimm21: Evidence that a site of N action differs from a site of N recognition

We report the isolation and characterization of a new mutation in the hybrid phage λimm21. Both genetic and physiological studies demonstrate that this new mutation, N_21?1, is similar to N mutations of phage λ. As in the case of the N gene of λ (Ni_λ), the N_21?1 mutation maps immediately to the left of the cI gene and has a pleiotropic effect on the expression of phage functions. Although these studies strongly suggest that phage 21 has an N function, they do not definitely locate the N_21?1 mutation within the N₂₁ structural gene.Reported here are studies demonstrating that N₂₁ acts in trans, similar to N_λ, to stimulate the expression of phage functions. N products show an immunity specificity; N₂₁ being only active on phage carrying the immunity region of phage 21, while the n_λ is only active on phage carrying the immunity region of λ or phage 434. However, one site of action for N_λ can be rescued from phage 21. We propose that the specificity of an N function is determined by its sites of recognition and that these sites may be different from the sites of N action.     

HLA 1990

The HLA system encompasses approximately one thousandth of the human genome and contains a series of closely linked loci coding for molecules which provide the context for the recognition of antigens by T lymphocytes. Within the HLA system, several phenotypically expressed and genomic polymorphisms can be distinguished. These polymorphisms are described and the main objectives for the future research are discussed.     

Further resolution of the quail karyotype and characterization of microchromosomes by counterstain-enhanced fluorescence

Sequential staining with a counterstain-contrasted fluorescent R-banding technique (chromomycin A3/distamycin A-DAPI) followed by DAPI-actinomycin D-induced quinacrine-fluorescence-Hoechst 33258 (QFH)-type banding allowed the identification of quail chromosomes up to chromosome 19. The chromomycin A3-positive staining behavior of the W chromosome and of the heterochromatic areas of most microchromosomes indicated their GC-rich nature.