Nonfunctional Tricarboxylic Acid Cycle and the Mechanism of Glutamate Biosynthesis in Acetobacter suboxydans

Acetobacter suboxydans does not contain an active tricarboxylic acid cycle, yet two pathways have been suggested for glutamate synthesis from acetate catalyzed by cell extracts: a partial tricarboxylic acid cycle following an initial condensation of oxalacetate and acetyl coenzyme A. and the citramalate-mesaconate pathway following an initial condensation of pyruvate and acetyl coenzyme A. To determine which pathway functions in growing cells, acetate-1-(14)C was added to a culture growing in minimal medium. After growth had ceased, cells were recovered and fractionated. Radioactive glutamate was isolated from the cellular protein fraction, and the position of the radioactive label was determined. Decarboxylation of the C5 carbon removed 100% of the radioactivity found in the purified glutamate fraction. These experiments establish that growing cells synthesize glutamate via a partial tricarboxylic acid cycle. Aspartate isolated from these hydrolysates was not radioactive, thus providing further evidence for the lack of a complete tricarboxylic acid cycle. When cell extracts were analyzed, activity of all tricarboxylic acid cycle enzymes, except succinate dehydrogenase, was demonstrated.     

Randomized trial on 14 versus 7 days of esomeprazole, moxifloxacin, and amoxicillin for second-line or rescue treatment of Helicobacter pylori infection

Background: Triple therapy with a proton pump inhibitor, moxifloxacin, and amoxicillin has been proven effective in first‐line treatment of Helicobacter pylori infection. Aim: To explore 1, the value of triple therapy with esomeprazole, moxifloxacin, and amoxicillin in second‐line or rescue treatment of Caucasian patients and 2, the impact of treatment duration on eradication success. Methods: H. pylori‐infected patients with at least one previous treatment failure were randomized to oral esomeprazole 20 mg b.i.d., moxifloxacin 400 mg o.d., and amoxicillin 1000 mg b.i.d. for either 7 (EMA‐7) or 14 days (EMA‐14). Eradication was confirmed by 13C urea breath test. Antimicrobial susceptibility testing was performed in all patients at baseline and in patients who failed treatment. Results: Eighty patients were randomized, and 60% had ≥2 previous treatment failures. Pretreatment resistance against clarithromycin and metronidazole was found in 70.5 and 61.5% of cases, respectively. The intention‐to‐treat eradication rate was significantly higher after EMA‐14 compared with EMA‐7 (95.0 vs 78.9%, p = .036). No independent risk factor for treatment failure could be identified. There were no serious adverse events. Five of the EMA‐14 patients (12.5%) compared with none of the EMA‐7 patients discontinued prematurely because of adverse events (p = .031). Post‐treatment resistance against moxifloxacin was found in one of seven patients with isolated organisms (14.3%). Conclusion: Second‐line/rescue H. pylori eradication therapy with esomeprazole, moxifloxacin, and amoxicillin is very effective and well tolerated. Fourteen days of treatment significantly increase the eradication rate but also the rate of adverse events.     

Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family

The GIY-YIG nuclease domain was originally identified in homing endonucleases and enzymes involved in DNA repair and recombination. Many of the GIY-YIG family enzymes are functional as monomers. We show here that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recognizes the symmetric sequence 5′-CCGC/GG-3′ (‘/’ indicates the cleavage site) is a tetramer in solution. Moreover, biochemical and kinetic studies provided here demonstrate that the Cfr42I tetramer is catalytically active only upon simultaneous binding of two copies of its recognition sequence. In that respect Cfr42I resembles the homotetrameric Type IIF restriction enzymes that belong to the distinct PD-(E/D)XK nuclease superfamily. Unlike the PD-(E/D)XK enzymes, the GIY-YIG nuclease Cfr42I accommodates an extremely wide selection of metal-ion cofactors, including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺, Cu²⁺ and Ca²⁺. To our knowledge, Cfr42I is the first tetrameric GIY-YIG family enzyme. Similar structural arrangement and phenotypes displayed by restriction enzymes of the PD-(E/D)XK and GIY-YIG nuclease families point to the functional significance of tetramerization.     

Do global change experiments overestimate impacts on terrestrial ecosystems?

In recent decades, many climate manipulation experiments have investigated biosphere responses to global change. These experiments typically examined effects of elevated atmospheric CO(2), warming or drought (driver variables) on ecosystem processes such as the carbon and water cycle (response variables). Because experiments are inevitably constrained in the number of driver variables tested simultaneously, as well as in time and space, a key question is how results are scaled up to predict net ecosystem responses. In this review, we argue that there might be a general trend for the magnitude of the responses to decline with higher-order interactions, longer time periods and larger spatial scales. This means that on average, both positive and negative global change impacts on the biosphere might be dampened more than previously assumed.     

Microbiome Influences Prenatal and Adult Microglia in a Sex-Specific Manner

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Ovine placenta at high altitudes: comparison of animals with different times of adaptation to hypoxic environment

Fetal growth and newborn weight from ovine gestations at high altitudes (HA) are greater in ewes that live at HA for several generations than in those native to low altitudes (LA) exposed to HA only during pregnancy. Because the placenta is a key regulator of fetal growth, the present study compared placental characteristics in term pregnancies among ewes native to HA and LA. Conception occurred at HA and ewes continued to reside at HA throughout pregnancy or conception occurred at LA and ewes were transported to HA or remained at LA (controls). Ewes native to LA were moved to HA shortly after mating (group LH) and joined with pregnant ewes native to HA (group HH). After parturition, placental cotyledons were counted and measured for total area and histological estimation of surface occupied by vasculature. The total surface of the cotyledons and surface occupied by vasculature were greater at HA, whereas the number of cotyledons was smaller at HA. These changes were more pronounced in ewes of the HH compared with the LH group. The present study showed that exposure to HA induces, in pregnant ewes, placental morphological changes that may improve maternal-fetal exchange. Moreover, because of accentuation of placental changes in ewes with long-term residence at HA, this appears to be an efficient mechanism of adaptation to hypobaric hypoxia.     

Structural and functional comparison of 15S- and 15R-specific cyclooxygenases from the coral Plexaura homomalla.

It has been known for 30 years that the gorgonian coral Plexaura homomalla contains either 15S- or 15R-configuration prostaglandins (PGs), depending on its location in the Caribbean. Recently we showed that the 15R-PGs in the R-variety of P. homomalla are formed by a unique cyclooxygenase (COX) with 15R oxygenation specificity [Valmsen, K., J?rving, I., Boeglin, W.E., Varvas, K., Koljak, R., Pehk, T., Brash, A.R. & Samel, N. (2001) Proc. Natl. Acad. Sci. USA98, 7700]. Here we describe the cloning and characterization of a closely related COX protein (97% amino acid sequence identity) from the S-variety of P. homomalla. Functional expression of the S-variant COX cDNA in Sf9 insect cells followed by incubation with exogenous arachidonic acid resulted in formation of PG products with > 98% 15S-configuration. Mutational analysis was performed on a suggested active site determinant of C-15 oxygenation specificity, position 349 (Val in all S-specific COX, Ile in 15R-COX). The 15S-COX Val349 to Ile mutant formed 35% 15R-PGs, while the reverse mutation in the 15R-COX (Ile349Val) led to formation of 70% 15S-products. This establishes position 349 as an important determinant of the product stereochemistry at C-15. Our characterization of the enzyme variants demonstrates that very minor sequence divergence accounts for the content of epimeric PGs in the two variants of P. homomalla and that the differences do not arise by isomerization of the products.