Marine Ammonia- and Nitrite-Oxidizing Bacteria: Serological Diversity Determined by Immunofluorescence in Culture and in the Environment

Immunofluorescence assays for marine ammonium- and nitrite-oxidizing bacteria were used to assess the diversity of nitrifying bacteria isolated from marine environments. The antisera show relatively broad specificity, in that each reacts with several strains of the same physiological type as the strain to which the antiserum was prepared. The antisera do not, however, react with any strains of differing physiological type. Seventy percent of the 30 unidentified ammonium-oxidizing isolates tested reacted with one or both of the antisera produced to marine ammonium-oxidizing strains, and 8 of the 9 unidentified nitrite-oxidizing strains tested reacted with 1 or more of the 3 nitrite oxidizer antisera used. Ammonium- and nitrite-oxidizing bacteria were enumerated in samples taken in a depth profile (to 750 m) in the Southern California Bight by immunofluorescence assays for two ammonium oxidizers and two nitrite oxidizers. Average abundances of the two types of nitrifiers were 3.5 × 10⁵ and 2.8 × 10⁵ cells liter⁻¹, respectively. Nitrifiers constitute 0.1 to 0.8% of the total bacterial population in these samples.     

Diel Production and Microheterotrophic Utilization of Dissolved Free Amino Acids in Waters Off Southern California

Diel patterns of dissolved free amino acid (DFAA) concentration and microheterotrophic utilization were examined in the spring and fall of 1981 in euphotic waters from the base of the mixed layer off the southern California coast. The average depths of the isotherms sampled were 19.2 m for spring and 9.0 m for fall. Total DFAA levels were generally higher in the spring than in the fall, 18 to 66 nM and 14 to 20 nM, respectively. Two daily concentration maxima and minima were observed for total DFAAs as well as for most individual DFAAs. Maxima were usually measured in the mid-dark period and in the early afternoon; minima were typically observed in early morning and late afternoon. Bacterial cell numbers reached maximal values near midnight in both seasons. These increases coincided with one of the total DFAA maxima. The second total DFAA maximum occurred in early to midafternoon, during the time of maximum photosynthetic carbon production and rapid dissolved amino acid utilization. Microbial metabolism (incorporation plus respiration) of selected ³H-amino acids was 2.7 to 4.1 times greater during the daylight hours. DFAA turnover times, based on these metabolic measurements, ranged between 11 and 36 h for the amino acids tested, and rates were 1.7 to 3.7 times faster in the daylight hours than at night. DFAA distributions were related to primary production and chlorophyll a concentrations. Amino acids were estimated to represent 9 to 45% of the total phytoplankton exudate. Microheterotrophic utilization or production of total protein amino acids was estimated as 3.6 μg of C liter⁻¹ day⁻¹ in spring and 1.9 μg of C liter⁻¹ day⁻¹ in the fall. Assimilation efficiency for dissolved amino acids averaged 65% for marine microheterotrophs.     

Manganese Tetraphenylporphyrins Catalyzed Selective Oxidation of Purine Derivatives

Abstract

The oxidation of purine derivatives using porphyrins as catalysts and dimethyldioxirane (DMDO) as oxygen atom donor is reported. The regioselectivity of the oxidation was found to be dependent on the presence of a free OH moiety on the N(9)-side chain of the substrate and on the structure of the catalyst.     

Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.     

Stability study of Prulifloxacin and Ulifloxacin in human plasma by HPLC–DAD

A new and specific HPLC–DAD method for the direct determination of Prulifloxacin and its active metabolite, Ulifloxacin, in human plasma has been developed. Plasma samples were analysed after a simple solid phase extraction (SPE) clean-up using a new HILIC stationary phase based high-performance liquid chromatography (HPLC) column and an ammonium acetate buffer (5?mM, pH 5.8)/acetonitrile (both with 1% Et₃N, v/v) mobile phase in isocratic elution mode, with Danofloxacin as the internal standard. Detection was performed using DAD from 200 to 500?nm and quantitative analyses were carried out at 278?nm. The LOQ of the method was 1?μg/mL of the cited analytes and the calibration curve showed a good linearity up to 25?μg/mL. For both analytes the precision (RSD%) and the trueness (bias%) of the method fulfil with International Guidelines. The method was applied for stability studies, at three QC concentration levels, in human plasma samples stored at different temperature of?+?25,?+?4 and ?20?°C in order to evaluate plasma stability profiles.     

Role of anti-cyclic citrullinated peptide antibodies in discriminating patients with rheumatoid arthritis from patients with chronic hepatitis C infection-associated polyarticular involvement

This study was performed to assess the utility of anti-cyclic citrullinated peptide (anti-CCP) antibodies in distinguishing between patients with rheumatoid arthritis (RA) and patients with polyarticular involvement associated with chronic hepatitis C virus (HCV) infection. Serum anti-CCP antibodies and rheumatoid factor (RF) were evaluated in 30 patients with RA, 8 patients with chronic HCV infection and associated articular involvement and 31 patients with chronic HCV infection without any joint involvement. In addition, we retrospectively analysed sera collected at the time of first visit in 10 patients originally presenting with symmetric polyarthritis and HCV and subsequently developing well-established RA. Anti-CCP antibodies and RF were detected by commercial second-generation anti-CCP2 enzyme-linked immunosorbent assay and immunonephelometry respectively. Anti-CCP antibodies were detected in 23 of 30 (76.6%) patients with RA but not in patients with chronic HCV infection irrespective of the presence of articular involvement. Conversely, RF was detected in 27 of 30 (90%) patients with RA, 3 of 8 (37.5%) patients with HCV-related arthropathy and 3 of 31 (9.7%) patients with HCV infection without joint involvement. Finally, anti-CCP antibodies were retrospectively detected in 6 of 10 (60%) patients with RA and HCV. This indicates that anti-CCP antibodies can be useful in discriminating patients with RA from patients with HCV-associated arthropathy.     

Essential role of A-kinase anchor protein 121 for cAMP signaling to mitochondria

A-Kinase anchor proteins (AKAPs) immobilize and concentrate protein kinase A (PKA) isoforms at specific subcellular compartments. Intracellular targeting of PKA holoenzyme elicits rapid and efficient phosphorylation of target proteins, thereby increasing sensitivity of downstream effectors to cAMP action. AKAP121 targets PKA to the cytoplasmic surface of mitochondria. Here we show that conditional expression of AKAP121 in PC12 cells selectively enhances cAMP.PKA signaling to mitochondria. AKAP121 induction stimulates PKA-dependent phosphorylation of the proapoptotic protein BAD at Ser(155), inhibits release of cytochrome c from mitochondria, and protects cells from apoptosis. An AKAP121 derivative mutant that localizes on mitochondria but does not bind PKA down-regulates PKA signaling to the mitochondria and promotes apoptosis. These findings indicate that PKA anchored by AKAP121 transduces cAMP signals to the mitochondria, and it may play an important role in mitochondrial physiology.     

Fatty acid composition of phospholipids, triglycerides and cholesterol in serum of castrated and estradiol treated rats

We have studied the levels of phospholipids, triglycerides, cholesterol esters, and their fatty acid composition in serum for normal, castrated and estradiol treated rats. The sex hormones did not greatly affect the levels of the various lipid fractions which did not undergo great significant variations, under the different treatments. More evident variations occurred in the percent composition of fatty acid and in the content of the various saturated (SAT), unsaturated (UNSAT), essential (EFA) and non essential fatty acids (NEFA). We studied the most important ratios: EFA/NEFA; UNS/SAT; 16:0/16:1; 18:0/18:1, 18:2/18:3; 18:2/20:4. 16:0/16:1; 18:0/18:1 represent the delta9 desaturase, one specific for palmitic, the other for stearic acid. 18:2/18:3 ratio is an index of the delta6 desaturase activity: 18:2/20:4 ratio of delta5 desaturase-elongase. Most changes were evident in triglycerides. We observed a different behaviour of the UNS/SAT and EFA/NEFA ratios in phospholipids and cholesterol esters, which may reflect either an effect of the sex hormones on the exchange of fatty acids between the same lipid fractions, or a redistribution of lipids among different tissues. Great variations were observed of the ratios 16:0/16:1; 18:0/18:1; 18:2/18:3; 18:2/20:4, which are ascribed a different effect of the sex hormones of delta9, delta6, delta5 desaturases.     

Study of <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release and Percutaneous Absorption of Fluconazole from Topical Dosage Forms

The present study aimed to evaluate different dosage forms, emulsions, emulgels, lipogels, and thickened microemulsion-based hydrogel, as fluconazole topical delivery systems with the purpose of determining a formulation with the capacity to deliver the whole active compound and maintain it within the skin so as to be considered a useful formulation either for topical mycosis treatment or as adjuvant in a combined therapy for Cutaneous Leishmaniasis. Propylene glycol and diethyleneglycol monoethyl ether were used for each dosage form as solvent for the drug and also as penetration enhancers. In vitro drug release after application of a clinically relevant dose of each formulation was evaluated and then microemulsions and lipogels were selected for the in vitro penetration and permeation study. Membranes of mixed cellulose esters and full-thickness pig ear skin were used for the in vitro studies. Candida albicans was used to test antifungal activity. A microemulsion containing diethyleneglycol monoethyl ether was found to be the optimum formulation as it was able to deliver the whole contained dose and enhance its skin penetration. Also this microemulsion showed the best performance in the antifungal activity test compared with the one containing propylene glycol. These results are according to previous reports of the advantages of microemulsions for topical administration and they are very promising for further clinical evaluation.