Isolation and characterization of extragenic mutations affecting the expression of the uroporphyrinogen decarboxylase gene (HEM12) in Sacharomyces cerevisiae

Uroporphyrinogen decarboxylase (Uro-d; EC 4.1.1.37), the fifth enzyme in the heme biosynthetic pathway, which catalyzes the sequential decarboxylation of uroporphyrinogen to coproporphyrinogen, is encoded by the HEM12 gene in Saccharomyces cerevisiae. The HEM12 gene is transcribed into a major short mRNA and a minor longer one, approximately 1.35 and 1.55 kb, respectively, in size, and that differ in the 5′ untranslated region. “Uroporphyric” mutants, which have no mutations in the HEM12 gene but accumulate uroporphyrinogen, a phenotype chracteristic of partial Uro-d deficiency, were investigated. Genetic analysis showed that the mutant phenotype depends on the combined action of two unlinked mutations, udt1 and either ipa1, ipa2, or ipa3. ipa1 is tightly linked to HEM12 The mutation udt1 apparently acts specifically on the HEM12 gene, and causes a six to tenfold decrease in the levels of the short HEM12 mRNA, in the β-galactosidase activity of a HEM12-lacZ fusion, in immunodetectable protein and enzyme activity. But heme synthesis is normal and porphyrin accumulation was modest. The mutations ipa1, ipa2, and ipa3 had no phenotype on their own, but they caused an increase in porphyrin accumulation in a udt1 background. This multiplicity of genetic factors leading to uroporphyric yeast cells closely resembles the situation in human porphyria cutanea tarda.     

Epr Studies on the Effects of Complexation of Heme by Hemopexin upon its Reactions with Organic Peroxides

Hemopexin, a heme-binding serum glycoprotein, is thought to play an important role in the prevention of oxidative damage that may be catalysed by free heme. Through the use of EPR techniques, the generation of free radicals from organic hydroperoxides by heme and heme-hemopexin complexes, and the concomitant formation of high oxidation-state iron species has been studied; these species are implicated as causative agents in processes such as cardiovascular disease and carcinogenesis. From the rates of production of these species from both n-alkyl and branched hydroperoxides, it has been inferred that the dramatic reduction in the yield of oxidising species generated by heme upon its complexation with hemopexin arises from steric hindrance of the access of hydroperoxide to the bound heme.     

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.     

The rfb locus from Pseudomonas aeruginosa strain PA103 promotes the expression of O antigen by both LPS-rough and LPS-smooth isolates from cystic fibrosis patients

Summary
Strains of Pseudomonas aeruginosa initially isolated from patients with cystic fibrosis (CF) often express a smooth lipopolysaccharide (LPS) containing many long O side-chain antigens, but once a chronic infection is established, strains recovered from these patients express little or no LPS O antigen. The genetic basis for this loss of O antigen expression by P. aeruginosa CF isolates is unknown. We report here that 20 CF isoiates of P. aeruginosa , 13 of which are LPS-rough, were each capable of expressing serogroup 011 antigen when provided with the rfb iocus from P. aeruginosa serogroup 011 strain PA103 on the recombinant plasmid pLPS2. Eight of the thirteen LPS-rough isolates co-expressed another, presumably endogenous, O antigen when they contained pLPS2. Different subcloned regions of pLPS2 complemented distinct strains to restore endogenous O antigen expression. These data suggest that the loss of O antigen expression by P. aeruginosa CF isolates results from alterations specific to the rfb region, and is not due to mutations involving other loci or ancillary LPS genes.     

Distribution of the enantiomers of hydroxychloroquine and its metabolites in ocular tissues of the rabbit after oral administration of racemic-hydroxychloroquine

The disposition of the enantiomers of hydroxychloroquine (HCQ) and its major metabolites in ocular tissues of rabbits has been studied. Both albino, New Zealand White (NZW), and pigmented animals were administered daily oral doses of rac-HCQ, (S)-HCQ or (R)-HCQ (20 mg/kg) over 1, 6, or 8 day periods or for 8 days followed by a 7-day washout period. At the end of the study periods, plasma and whole blood samples were collected and the rabbits were sacrificed. The eyes were collected, the aqueous humor removed with a syringe, and the eyes separated into the cornea, lens, vitreous body, iris, choroid-retina, sclera, and conjunctiva. The concentrations of (R)-HCQ, (S)-HCQ, and their respective metabolites were determined using a validated enantioselective liquid chromatographic assay. The data from these studies indicate that HCQ accumulated in both pigmented and nonpigmented ocular tissues. In the pigmented tissues, HCQ and its metabolites were bound to melanin and the binding was not enantiospecific. In the nonpigmented tissues and in the iris and retina-choroid of the NZW rabbits, the accumulation appeared to be the result of a reversible and enantioselective binding of HCQ and its metabolites to an unidentified biopolymer present in these ocular tissues. © 1994 Wiley-liss, Inc.     

Influences of sea level changes and volcanic eruptions on Holocene vegetation in Tonga

Here, we investigate Mid- to Late-Holocene vegetation changes in low-lying coastal areas in Tonga and how changing sea levels and recurrent volcanic eruptions have influenced vegetation dynamics on four islands of the Tongan archipelago (South Pacific). To investigate past vegetation and environmental change at Ngofe Marsh (‘Uta Vava’u), we examined palynomorphs (pollen and spores), charcoal (fire), and sediment characteristics (volcanic activity) from a 6.7-m-long sediment core. Radiocarbon dating indicated the sediments were deposited over the last 7700 years. We integrated the Ngofe Marsh data with similar previously published data from Avai’o’vuna Swamp on Pangaimotu Island, Lotofoa Swamp on Foa Island, and Finemui Swamp on Ha’afeva Island. Plant taxa were categorized as littoral, mangrove, rainforest, successional/ disturbance, and wetland groups, and linear models were used to examine relationships between vegetation, relative sea level change, and volcanic eruptions (tephra). We found that relative sea level change has impacted vegetation on three of the four islands investigated. Volcanic eruptions were not identified as a driver of vegetation change. Rainforest decline does not appear to be driven by sea level changes or volcanic eruptions. From all sites analyzed, vegetation at Finemui Swamp was most sensitive to changes in relative sea level. While vegetation on low-lying Pacific islands is sensitive to changing sea levels, island characteristics, such as area and elevation, are also likely to be important factors that mediate specific island responses to drivers of change.     

CHIRBASE,a graphical molecular database on the separation of enantiomers by liquid-, supercritical fluid-, and gas chromatography

In order to cope with the increasing number of publications on the separation of enantiomers by chromatography on a chiral stationary phase, the graphical molecular database CHIRBASE was created. In the present state, the database package covers information (structural, bibiographic, and chromatographic data) on liquid-, supercritical fluid-, and gas chromatography; other methods will follow. CHIRBASE, running on the MDL software Chembase®, meets the requirements of contemporary information management in the chemical and pharmaceutical industry. (Detailed information including a demo-version of each part of CHIRBASE can be obtained from the authors on request.) © 1993 Wiley-Liss, Inc.