Ultrahigh resolution structures of nitrophorin 4: heme distortion in ferrous CO and NO complexes

Nitrophorin 4 (NP4), a nitric oxide (NO)-transport protein from the blood-sucking insect Rhodnius prolixus, uses a ferric (Fe3+) heme to deliver NO to its victims. NO binding to NP4 induces a large conformational change and complete desolvation of the distal pocket. The heme is markedly nonplanar, displaying a ruffling distortion postulated to contribute to stabilization of the ferric iron. Here, we report the ferrous (Fe2+) complexes of NP4 with NO, CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy, flash photolysis, and ultrahigh-resolution crystallography (resolutions vary from 0.9 to 1.08 A). The absorption spectra, both in solution and in the crystal, are typical for six-coordinated ferrous complexes. Closure and desolvation of the distal pocket occurs upon binding CO or NO to the iron regardless of the heme oxidation state, confirming that the conformational change is driven by distal ligand polarity. The degree of heme ruffling is coupled to the nature of the ligand and the iron oxidation state in the following order: (Fe3+)-NO > (Fe2+)-NO > (Fe2+)-CO > (Fe3+)-H2O > (Fe2+)-H2O. The ferrous coordination geometry is as expected, except for the proximal histidine bond, which is shorter than typically found in model compounds. These data are consistent with heme ruffling and coordination geometry serving to stabilize the ferric state of the nitrophorins, a requirement for their physiological function. Possible roles for heme distortion and NO bending in heme protein function are discussed.     

Strain-specific ureolytic microbial calcium carbonate precipitation

During a study of ureolytic microbial calcium carbonate (CaCO(3)) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO(3) crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K(m)) and maximum hydrolysis rates (V(max)) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.     

Increased serum IgM antibodies directed against phosphatidyl inositol (Pi) in chronic fatigue syndrome (CFS) and major depression: evidence that an IgM-mediated immune response against Pi is one factor underpinning the comorbidity between both CFS and depression

Major depression and chronic fatigue syndrome (CFS) are accompanied by signs of oxidative and nitrosative stress (O&NS) and an inflammatory response. Phosphatidyl inositol (Pi) is thought to play a role in depression. The aim of the present study is to examine whether depression and CFS are characterized by an IgM-mediated immune response directed against Pi. Toward this end, this study examines the serum IgM antibodies directed against Pi in 14 patients with major depression, 14 patients with CFS, 14 subjects with partial CFS, and in 11 normal controls. We found that the prevalence and mean value for the serum IgM levels directed against Pi were significantly greater in patients with major depression and CFS than in normal controls and patients with partial CFS. There were significant and positive correlations between serum IgM levels directed against Pi and two symptoms of the FibroFatigue Scale, i.e. fatigue and depression. The results show that an IgM-related immune response directed against Pi may occur in both depression and CFS and may play a role in the pathophysiology of the key symptom of CFS and major depression. It is suggested that the above disorders in Pi result from increased O&NS in both depression and CFS. Autoanti-Pi antibodies may have biological effects, for example, by changing inositol 1,4,5-triphosphate (IP3), phosphatidylinositol-4,5-bisphosphate (PIP2), diacylglycerol and phosphatidylinositol-3,4,5-triphosphate (PIP3) production, thus interfering with intracellular signalling processes. Future research in major depression and CFS should focus on the functional consequences of the immune responses directed against Pi.     

Development and evolution of adaptive polyphenisms

Phenotypic plasticity is the primitive character state for most if not all traits. Insofar as developmental and physiological processes obey the laws of chemistry and physics, they will be sensitive to such environmental variables as temperature, nutrient supply, ionic environment, and the availability of various macro- and micronutrients. Depending on the effect this phenotypic plasticity has on fitness, evolution may proceed to select either for mechanisms that buffer or canalize the phenotype against relevant environmental variation or for a modified plastic response in which some ranges of the phenotypic variation are adaptive to particular environments. Phenotypic plasticity can be continuous, in which case it is called a reaction norm, or discontinuous, in which case it is called a polyphenism. Although the morphological discontinuity of some polyphenisms is produced by discrete developmental switches, most polyphenisms are due to discontinuities in the environment that induce only portions of what is in reality a continuous reaction norm. In insect polyphenisms, the environmental variable that induces the alternative phenotype is a token stimulus that serves as a predictor of, but is not itself, the environment to which the polyphenism is an adaptation. In all cases studied so far, the environmental stimulus alters the endocrine mechanism of metamorphosis by altering either the pattern of hormone secretion or the pattern of hormone sensitivity in different tissues. Such changes in the patterns of endocrine interactions result in the execution of alternative developmental pathways. The spatial and temporal compartmentalization of endocrine interactions has produced a developmental mechanism that enables substantial localized changes in morphology that remain well integrated into the structure and function of the organism.     

Structural analysis of the oligosaccharide alditols released from the jelly coat of Rana dalmatina eggs by reductive beta-elimination

A combination of ion-exchange chromatography and high performance liquid chromatography (HPLC) has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of oviducal mucins obtained from the jelly coat of Rana dalmatina. The primary structures of 26 O-glycans were determined by one-dimensional and two-dimensional 1H and 1H13C NMR spectroscopy. As observed for 20 other amphibian species, these carbohydrate chains are highly species-specific. The main typical feature of the species R. dalmatina consists in the presence of the backbone Gal(beta1-3)[Gal(beta1-4)]Gal(beta1-3)GalNAc-ol, previously observed among Ranidae, such as R. temporaria and R. ridibunda. Nevertheless, the nature of carbohydrates present at the periphery of the glycans perfectly differentiates the three species.     

Synthesis and evaluation of the quorum sensing inhibitory effect of substituted triazolyldihydrofuranones

Acylhomoserine lactone (AHL) analogues in which the amide function is replaced by a triazole group were synthesized and evaluated for their effect on quorum sensing (QS) and biofilm formation in Burkholderia cenocepacia and Pseudomonas aeruginosa. In addition, the influence of the length of the acyl-mimicking chain was investigated. The compounds showed selectivity between two different AHL QS systems. 3-(1H-1,2,3-Triazol-1-yl)dihydrofuran-2(3H)-ones, in which the 4-substituent best resembled the acyl chain of the native AHL molecule exhibited significant QS agonistic and antagonistic activities. Replacing this aliphatic substituent by a phenyl-containing moiety resulted in active inhibitors of QS. The most active compounds showed biofilm inhibitory as well as biofilm eradicating activities in both test organisms.     

Clustering of lipid-bound annexin V may explain its anticoagulant effect.

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.