Spectroscopic and mutational analysis of the blue-light photoreceptor AppA: a novel photocycle involving flavin stacking with an aromatic amino acid

The flavoprotein AppA is a blue-light photoreceptor that functions as an antirepressor of photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides. Heterologous expression studies show that FAD binds to a 156 amino acid N-terminal domain of AppA and that this domain is itself photoactive. A pulse of white light causes FAD absorption to be red shifted in a biphasic process with a fast phase occurring in <1 micros and a slow phase occurring at approximately 5 ms. The absorbance shift was spontaneously restored over a 30 min period, also in a biphasic process as assayed by fluorescence quenching and electronic absorption analyses. Site-directed replacement of Tyr21 with Leu or Phe abolished the photochemical reaction implicating involvement of Tyr21 in the photocycle. Nuclear magnetic resonance analysis of wild-type and mutant proteins also indicates that Tyr21 forms pi-pi stacking interactions with the isoalloxazine ring of FAD. We propose that photochemical excitation of the flavin results in strengthening of a hydrogen bond between the flavin and Tyr 21 leading to a stable local conformational change in AppA.     

Is nitric oxide luteolytic or antiluteolytic?

Nitric oxide (NO) has been reported to be luteolytic based on treatment of cows in vivo with an inhibitor of nitric oxide synthase (NOS-produces NO), which delayed the decline in progesterone by two to three days [Jaroszewki J, Hansel, W. Intraluteal administration of a nitric oxide synthase blocker stimulates progesterone, oxytocin secretion and prolongs the life span of the bovine corpus luteum. Proc Soc Exptl Biol Med 2000;224:50-5; Skarzynski D, Jaroszewki J, Bah, M, et al. Administration of nitric oxide synthase inhibitor counteracts prostaglandin F(2alpha)-induced luteolysis in cattle. Biol Reprod 2003;68:1674-81]. The objective of this experiment was to determine the effect of a long acting NO donor or a NOS inhibitor infused chronically into the interstitial tissue of the ovarian vascular pedicle adjacent to the ovary with a corpus luteum on secretion of progesterone during the ovine estrous cycle. Ewes were treated either with Vehicle (N=5); Diethylenetriamine (DETA-control for DETA-NONOate; N=5); (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NONOate-long acting NO donor; N=6); or l-nitro-arginine methyl ester (l-NAME-NOS inhibitor; N=6) every 6 h from 24:00 h (0 h) on day 8 through 18:00 h on day 18 of the estrous cycle. Jugular venous blood was collected every 6h for analysis for progesterone and corpora lutea were collected at 18:00 h on day 18 and weighed. Weights of corpora lutea were heavier (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes, which did not differ amongst each other (P> or =0.05). Profiles of progesterone in jugular venous blood on days 8-18 differed (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes did not differ (P> or =0.05) amongst each other. It is concluded that NO is not luteolytic during the ovine estrous cycle, but may instead be antiluteolytic and prevent luteolysis.     

<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> induces aponecrosis in human aortic smooth muscle cells

Background  

The intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis.     

Potential regrowth and recolonization of salmonellae and indicators in biosolids and biosolid-amended soil

This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.     

Fate of air emboli in the pulmonary circulation

The lung serves an important nonrespiratory function by trapping and excreting venous air emboli. The site of trapping and the mechanism of excretion, however, are uncertain. To observe the behavior of bubbles in the pulmonary circulation, we injected venous air emboli into anesthetized dogs and videotaped their elimination from the pulmonary microcirculation by using in vivo microscopy. Small intravenous bubbles lodged exclusively in pulmonary arterioles and were eliminated from that site. To determine whether the gas was dissolving into nearby blood and then was carried to the capillaries for excretion, the rate of bubble radius change was measured during nonperfused conditions produced by balloon occlusion of lobar blood flow and compared with perfused conditions. Bubble volume decreased at the same rate during perfused and nonperfused conditions and thus was independent of regional blood flow. Molecular diffusion of gas directly across the arteriolar wall into alveolar spaces was the most likely mechanism of elimination because calculations based on the Fick equation for molecular diffusion predict an elimination rate nearly identical with those observed experimentally.     

Fine needle aspiration cytologic features of hepatic metastasis of granulosa cell tumor of the ovary. Differential diagnosis

The cytologic findings from a fine needle aspiration biopsy of hepatic metastases of a granulosa cell tumor are described. While the cytologic features of the tumor were characteristic, the early recurrence in an unusual site makes this case noteworthy. The differential diagnosis of granulosa cell tumors from other metastatic and primary liver tumors is discussed.     

Effects of short-term thermal stress on the plasma biochemical profiles of two Antarctic nototheniid species

Thermal elevation records in the Austral Ocean have raised questions about the physiological impacts on Antarctic organisms which have evolved under cold and stable water temperatures. Some notothenioid fishes exhibit species-specific responses to elevated temperature, yet the mechanisms involved in restoring homeostasis are unclear. Our study focused on the physiological effects of short-term (2–144 h) exposure to water temperatures of 8 °C on the plasma biochemical profiles of Notothenia coriiceps and Notothenia rossii, species that are abundant in Admiralty Bay, King George Island, Antarctic Peninsula, a region where increasing temperatures have been detected. Despite being phylogenetically similar, these species responded differently to thermal stress. N. rossii showed no changes in cortisol levels, and transient hyperglycemia was likely triggered by elevated catecholamine levels; conversely, metabolic and antioxidant defense parameters were unaffected. Increased gill Na⁺/K⁺-ATPase activity was observed only in N. rossii after 24 h at 8 °C, which assists in maintaining ionic homeostasis. In N. coriiceps, cortisol accurately indicated thermal stress. Increased cortisol levels in N. coriiceps additionally resulted in transient secondary responses such as hyperglycemia and hyperlactemia, as well as reduced levels of total protein, globulins and triglycerides. Unlike in N. rossii, catalase activity in N. coriiceps was modulated at 8 °C, and this parameter is thus considered a good biomarker of thermal stress. Results suggest that N. coriiceps is more sensitive to thermal stress than is N. rossii and that the former is a potential bioindicator for Admiralty Bay.     

Competition for energy between phosphoenolpyruvate and citrulline synthesis in guinea-pig liver mitochondria

1. The interrelationship between citrulline synthesis and phosphoenolpyruvate formation has been studied in guinea-pig liver mitochondria incubated with glutamate in State 3 and in the presence of uncoupler and oligomycin.

2. In coupled mitochondria the rate of phosphoenolpyruvate production was limited by a higher capacity of aspartate aminotransferase than that of phosphopyruvate carboxylase for the intramitochondrial oxalacetate. Citrulline formation was low due to the small production of NH₃ since glutamate oxidation in State 3 proceeds via the transamination pathway.

3. Inhibition of aspartate aminotransferase by aminooxyacetate in State 3 resulted in increases in both phosphoenolpyruvate and citrulline synthesis. Under uncoupled conditions, however, an increase of phosphoenolpyruvate formation was accompanied by a decrease of both citrulline production and the ATP content of the incubation medium. Restoration of the citrulline production was observed on the addition of exogenous ATP.

4. The results indicate that when energy is generated via substrate-level phosphorylation, the inhibition of citrulline production is probably due to a higher availability of GTP to the phosphopyruvate carboxylase than to the nucleoside diphosphate kinase.