Identification of a Two-Component Regulatory Pathway Essential for Mn(II) Oxidation in Pseudomonas putida GB-1

Bacterial manganese(II) oxidation has a profound impact on the biogeochemical cycling of Mn and the availability of the trace metals adsorbed to the surfaces of solid Mn(III, IV) oxides. The Mn(II) oxidase enzyme was tentatively identified in Pseudomonas putida GB-1 via transposon mutagenesis: the mutant strain GB-1-007, which fails to oxidize Mn(II), harbors a transposon insertion in the gene cumA. cumA encodes a putative multicopper oxidase (MCO), a class of enzymes implicated in Mn(II) oxidation in other bacterial species. However, we show here that an in-frame deletion of cumA did not affect Mn(II) oxidation. Through complementation analysis of the oxidation defect in GB-1-007 with a cosmid library and subsequent sequencing of candidate genes we show the causative mutation to be a frameshift within the mnxS1 gene that encodes a putative sensor histidine kinase. The frameshift mutation results in a truncated protein lacking the kinase domain. Multicopy expression of mnxS1 restored Mn(II) oxidation to GB-1-007 and in-frame deletion of mnxS1 resulted in a loss of oxidation in the wild-type strain. These results clearly demonstrated that the oxidation defect of GB-1-007 is due to disruption of mnxS1, not cumA::Tn5, and that CumA is not the Mn(II) oxidase. mnxS1 is located upstream of a second sensor histidine kinase gene, mnxS2, and a response regulator gene, mnxR. In-frame deletions of each of these genes also led to the loss of Mn(II) oxidation. Therefore, we conclude that the MnxS1/MnxS2/MnxR two-component regulatory pathway is essential for Mn(II) oxidation in P. putida GB-1.In living cells, manganese (Mn) is an essential trace element, required for enzymes such as superoxide dismutase and in photosystem II (7). In the environment, Mn cycles between a soluble reduced form [Mn(II)] and an insoluble oxidized form [Mn(III, IV)] that can adsorb other trace metals from the environment and serve as potent oxidizing agents. Thus, redox cycling of Mn has a profound effect on the bioavailability and geochemical cycling of many essential or toxic elements (40). Microorganisms, particularly bacteria, are capable of catalyzing the oxidation of Mn(II), thereby increasing the rate of formation of Mn(III, IV) by several orders of magnitude (39). Since Mn(III, IV) oxides are able to bind trace metals, the bacteria that catalyze their formation are good candidates for bioremediation of heavy metal contaminated sites (26, 39).Although bacterial Mn(II) oxidation is widespread, little is known about the physiological function of oxidation (40). The oxidation of Mn(II) to Mn(III) or Mn(IV) is thermodynamically favorable; thus, bacteria may derive energy from this reaction, although this has never been unequivocally proven (40). In addition, Mn(II) oxidation could protect cells from reactive oxygen species (4) or UV irradiation (11). Since oxidation occurs on the cell surface, the bacteria become coated with the solid Mn(IV) oxides, which may also provide protection from toxic heavy metals, predation, or phage infection (40). As a strong oxidant, Mn(IV) oxides could allow the bacteria to degrade refractory organic matter to low-molecular-weight compounds that could then be used to support bacterial growth (38). Conversely, Mn(II) oxidation may be a side reaction or the result of nonspecific interactions with cellular products (15). Identifying signals or conditions that regulate oxidation could provide some insight into the role of Mn(II) oxidation in the cell. Aside from a requirement for oxygen (28) and iron (27, 30), as well as the observation that oxidation occurs in stationary phase (23), very little is known about this regulation.The enzymes responsible for Mn(II) oxidation have been tentatively identified from some species of bacteria and in several cases the enzyme is a putative multicopper oxidase (MCO). MCOs are a family of enzymes that use four Cu ion cofactors to catalyze oxidation of diverse substrates such as metals and organic compounds (33). This family of enzymes is found in plants and fungi (laccase) and humans (ceruloplasmin), as well as in bacteria (35). Some fungi have been shown to use a laccase enzyme to oxidize Mn(II) (20). In both Leptothrix discophora SS-1 and Pedomicrobium sp. strain ACM 3067, the Mn(II)-oxidizing MCO was identified genetically (mofA [10] and moxA [31], respectively). A third MCO—MnxG—was identified both biochemically and genetically as the Mn(II) oxidase in Bacillus sp. strain SG-1 and related strains (14, 43). Recent work with the Mn(II)-oxidizing alphaproteobacterium Aurantimonas manganoxydans SI85-9A1 and Erythrobacter sp. strain SD21 has identified a second class of enzyme involved in Mn(II) oxidation: the heme-binding peroxidase named MopA (3). This class of enzyme had previously been shown to be used by fungi to oxidize Mn(II) (29), in some cases in concert with an MCO (34).Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium (9) whose genetic tractability and ease of growth under standard laboratory conditions make it an ideal model system for studying the physiology and mechanism of Mn(II) oxidation. Consequently, several random transposon mutagenesis screens have been undertaken with this organism to identify genes required for Mn(II) oxidation. These screens have identified several categories of genes as important for oxidation or the export of the oxidase to the cell surface: the ccm operon of c-type cytochrome synthesis genes (8, 13), genes encoding components of the trichloroacetic acid (TCA) cycle and the tryptophan biosynthesis pathway (8) and genes encoding a general secretory pathway (12). The Mn(II) oxidation-defective mutant GB-1-007 has a transposon insertion in the gene cumA that encodes a putative MCO (6). Therefore, P. putida GB-1 has been thought to use a similar mechanism as L. discophora SS-1, Pedomicrobium sp. strain ACM 3067, and Bacillus sp. to oxidize Mn(II).Because the available data suggested that CumA was an MCO essential for Mn(II) oxidation, we wanted to study its function in greater detail. We were hampered in this, however, by the fact that the transposon insertion in cumA resulted in a growth defect due to its polar effect on expression of the downstream cumB gene (6). In order to assess the role of CumA in Mn(II) oxidation without the complications arising from polarity, we generated an in-frame deletion of cumA and tested the ability of the resulting ΔcumA strain to form Mn(IV) oxides. Our results showed that cumA is dispensable for Mn(II) oxidation and have instead revealed a complex two-component regulatory pathway essential for Mn(II) oxidation in P. putida GB-1.     

Latent structure of Raven's Colored Progressive Matrices

The main goal of the study was to determine the constructive validity of Raven's Colored Progressive Matrices by means of item factor analysis. The most important topic within this objective is to determine the test dimensionality, since many authors report on finding several significant primary factors. The study included 2334 children aged 4 to 11. Several types of factor analysis were used in order to obviate the influence of technique on the results. Our data suggested 3 or 4 first order factors. Based on the usual McDonald definition, the primary factors obtained could be considered difficulty factors in the majority of cases. The necessary number of factors on age subsamples, extracted by parallel analysis, was between 3 and 5. Factor structure on age subsamples indicated the youngest ages, 4 and 5, to be essentially different from the older ones. This difference was identified as underdevelopment of the goal management mechanisms.     

Proliferation and motility of HaCaT keratinocyte derivatives is enhanced by fibroblast nemosis

The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.     

Psychological pain treatment in fibromyalgia syndrome: efficacy of operant behavioural and cognitive behavioural treatments

The present study focused on the evaluation of the effects of operant behavioural (OBT) and cognitive behavioural (CBT) treatments for fibromyalgia syndrome (FMS). One hundred and twenty-five patients who fulfilled the American College of Rheumatology criteria for FMS were randomly assigned to OBT (n = 43), CBT (n = 42), or an attention-placebo (AP) treatment (n = 40) that consisted of discussions of FMS-related problems. Assessments of physical functioning, pain, affective distress, and cognitive and behavioural variables were performed pre-treatment and post-treatment as well as 6 and 12 months post-treatment. Patients receiving the OBT or CBT reported a significant reduction in pain intensity post-treatment (all Fs > 3.89, all Ps < 0.01). In addition, the CBT group reported statistically significant improvements in cognitive (all Fs > 7.95, all P < 0.01) and affective variables (all Fs > 2.99, all Ps < 0.02), and the OBT group demonstrated statistically significant improvements in physical functioning and behavioural variables (all Fs > 5.99, all Ps < 0.001) compared with AP. The AP group reported no significant improvement but actually deterioration in the outcome variables. The post-treatment effects for the OBT and CBT groups were maintained at both the 6- and 12-month follow-ups. These results suggest that both OBT and CBT are effective in treating patients with FMS with some differences in the outcome measures specifically targeted by the individual treatments compared with an unstructured discussion group. The AP group showed that unstructured discussion of FMS-related problems may be detrimental.     

Mechanistic study of HCV polymerase inhibitors at individual steps of the polymerization reaction

Little is known about the mechanism of HCV polymerase-catalyzed nucleotide incorporation and the individual steps employed by this enzyme during a catalytic cycle. In this paper, we applied various biochemical tools and examined the mechanism of polymerase catalysis. We found that formation of a productive RNA-enzyme complex is the slowest step followed by RNA dissociation and initiation of primer strand synthesis. Various groups have reported several classes of small molecule inhibitors of hepatitis C virus NS5B polymerase; however, the mechanism of inhibition for many of these inhibitors is not clear. We undertook a series of detailed mechanistic studies to characterize the mechanisms of inhibition for these HCV polymerase inhibitors. We found that the diketoacid derivatives competitively bind to the elongation NTP pocket in the active site and inhibit both the initiation and elongation steps of polymerization. While both benzimidazoles and benzothiadiazines are noncompetitive with respect to the active site elongation NTP pocket, benzothiadiazine compounds competitively bind to the initiation pocket in the active site and inhibit only the initiation step of de novo RNA polymerization. The benzimidazoles bind to the thumb allosteric pocket and inhibit the conformational changes during RNA synthesis. We also observed a cross interaction between the thumb allosteric pocket and the initiation pocket using inhibitor-inhibitor cross competition studies. This information will be very important in designing combination therapies using two small molecule drugs to treat hepatitis C virus.     

Synthesis, antiviral activity, and pharmacokinetic evaluation of P3 pyridylmethyl analogs of oximinoarylsulfonyl HIV-1 protease inhibitors

As a continuation of the recently communicated discovery of oximinoarylsulfonamides as potent inhibitors of HIV-1 aspartyl protease, compounds bearing pyridylmethyl substituents at P3 were designed and synthesized. Potent analogs in this series provided low single-digit nanomolar EC50 values against both wild-type HIV and resistant mutant virus (A17), attenuated some 3- to 12-fold in the presence of 50% human serum. Pharmacokinetic results for compounds in this series showed good to excellent exposure when co-administered orally with an equal amount of ritonavir (5mg/kg each) in the rat, with average AUC >8 microg h/mL. Similar dosing in dog resulted in significantly lower plasma levels (average AUC <2 microg h/mL). The 3-pyridylmethyl analog 30 gave the best overall exposure (rat AUC=7.1 microg h/mL and dog AUC=4.9 microg h/mL), however, this compound was found to be a potent inhibitor of cytochrome P450 3A (Ki=2.4 nM).     

The impact of cognitive processors and conative regulators on specific motor abilities in boxers

The aim of the study was to analyze the relations of cognitive processors and conative regulators with specific motor abilities of elite boxers. Three sets of variables including 3 cognitive and 6 conative variables as predictors, and 6 specific motor (boxing) variables as criteria were used in a sample of 92 boxers. A series of regression analyses between the set of cognitive variables and particular criterion variables revealed a predominant impact of serial processor on specific motor abilities based primarily on specific speed (frequency of boxing technique performance). The series of regression analyses also showed a predominant negative impact of dysregulation of the organ function regulators from the set of conative variables on the manifestation of specific motor abilities in boxers. The data obtained in the study were used to develop an alternative model of the motor - cognitive - conative processes in boxing.     

Biomotor systems in elite junior judoists

The aim of the study was to assess the impact of motor abilities and morphological characteristics on junior judoka performance. A set of 14 morphological parameters and a set of 14 motor tests as predictor variables, and 3 variables evaluating judo performance as criteria were applied in a sample of 40 judoists aged 17 years +/-6 months. Three factors were isolated by factor analysis in morphological area: factor of muscle mass and bone volume (muscle and bone mass mesoectomorphy), factor of longitudinal skeleton dimensionality, and factor of subcutaneous adipose tissue (endomorphy). Four factors were isolated by factor analysis in motor area: factor of coordination and strength (regulated force), factor of movement frequency (speed), factor of muscular and cardiovascular endurance (endurance), and factor of tonus regulation and synergy regulation (flexibility/balance). Canonical correlation analysis between latent morphological and motor variables, and variables for assessment of competitive performance of junior judoists yielded two linear combinations, i.e. two pairs of canonical factors. Correlation in the first pair of canonical factors was underlain by the favorable impact of coordination/strength, speed, flexibility and balance, along with above-average muscle mass and bone volume, and above-average skeleton longitudinality on performance in judo. Correlation in the second pair of canonical factors was based on positive determination of above-average endurance along with moderate coordination/strength and speed, and below-average muscle mass and bone volume and skeleton longitudinality upon judo performance as expressed by the fight winning score.