ESEEM studies of substrate water and small alcohol binding to the oxygen-evolving complex of photosystem II during functional turnover

We report the first examination of exchangeable proton and MeOH interactions with the Mn catalytic cluster in photosystem II, under functional flash turnover conditions, using 2H ESEEM spectroscopy on the S2 and S0 multiline states. Deuterium-labeled water (D2O) and methyl d3-labeled methanol (DMeOH) are employed. It was discovered that a hyperfine resolved multiline S0 signal could be seen in the presence of D2O, the hyperfine structure of which depended on the presence or absence of methanol (MeOH). In the presence of DMeOH, significant dipolar coupling of the three methyl deuterons to the multiline centers in the S2 and S0 states was seen (S2, 0.65, 0.39(2) MHz; and S0, 0.60, 0.37(2) MHz). These are consistent with direct binding of the methoxy fragment to Mn. Assuming terminal Mn-OMe ligation, the couplings indicated a spin projection coefficient (rho) magnitude of approximately 2 for the ligating Mn in both the S2 and S0 states, with inferred Mn-O distances of approximately 1.9-2.0 A. In the presence of D2O, four classes of exchangeable deuterons were identified by ESEEM in S2 and S0. Three of these classes (1, 2, and 4) exhibited populations and coupling strengths that were essentially constant under various conditions of sample preparation, illumination turnover, and small alcohol addition. Class 3 could be modeled with constant coupling but a highly variable deuteron population (n3 approximately 0-10) depending in part on the preparation used. For all classes, the coupling parameters were very similar in S2 and S0. The favored interpretation is that the two strongest coupling classes (1 and 2) represent close binding of one water molecule to a single Mn which has an oxidation state of II in S0 and III in S2, and rho approximately 2 in both cases. This water is not displaced by MeOH, but either the water or MeOH is singly deprotonated upon MeOH binding. Class 4 represents approximately 2 water molecules which are not closely bound to Mn (Mn-deuteron distances of approximately 3.7-4.7 A). Class 3 probably represents protein matrix protons within approximately 4 A of the Mn in the cluster, which can be variably exchanged in different preparations.     

Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1

PKCdelta has been shown to be activated by insulin and to interact with insulin receptor and IRS. PKB(Akt) plays an important role in glucose transport and glycogen synthesis. In this study, we investigated the possibility that PKCdelta may be involved in insulin-induced activation of PKB. Studies were conducted on primary cultures of rat skeletal muscle. PKB was activated by insulin stimulation within 5min and reached a peak by 15-30min. Insulin also increased the physical association between PKCdelta with PKB and with PDK1. The insulin-induced PKCdelta-PKB association was PI3K dependent. PKB-PKCdelta association was accounted for by the involvement of PDK1. Overexpression of dominant negative PKCdelta abrogated insulin-induced association of PKCdelta with both PKB and PDK1. Blockade of PKCdelta also decreased insulin-induced Thr308 PKB phosphorylation and PKB translocation. Moreover, PKCdelta inhibition reduced insulin-induced GSK3 phosphorylation. The results indicate that insulin-activated PKCdelta interacts with PDK1 to regulate PKB.     

A novel meta-cleavage product hydrolase from Flavobacterium sp. ATCC27551

The organophosphate degrading (opd) gene cluster of plasmid pPDL2 of Flavobacterium sp. ATCC27551 contains a novel open-reading frame, orf243. This was predicted to encode an alpha/beta hydrolase distantly related to the meta-fission product (MFP) hydrolases such as XylF, PhnD, and CumD. By homology modeling Orf243 has most of the structural features of MFP hydrolases including the characteristic active site catalytic triad. The purified protein (designated MfhA) is a homotetramer and shows similar affinity for 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD), 2-hydroxymuconic semialdehyde (HMSA), and 2-hydroxy-5-methylmuconic semialdehyde (HMMSA), the meta-fission products of 3-methyl catechol, catechol, and 4-methyl catechol. The unique catalytic properties of MfhA and the presence near its structural gene of cis-elements required for transposition suggest that mfhA has evolved towards encoding a common hydrolase that can act on meta-fission products containing either aldehyde or ketone groups.     

Reduction and enhancement of Plasmodium falciparum transmission by endemic human sera

Transmission of Plasmodium falciparum from man to mosquito can be affected by human sera. Whereas serum-dependent reduction of transmission has been shown to be reproducible, there is limited evidence for enhancement of transmission. We aimed to assess the prevalence and reproducibility of transmission enhancement (TE) by human sera from different geographic areas (n = 642), in comparison with the capacity for transmission reduction (TR). The overall prevalence of TE (7%) was lower than that of TR (48%) and its effect generally weaker but reproducible in repeated measurements. TR but not TE showed a significant association with the presence of serum antibodies against Pfs48/45 and a non-significant trend to the presence of anti-Pfs230 antibodies.     

Application of mass spectrometry to the discovery of biomarkers for detection of prostate cancer

There has been an impressive emergence of mass spectrometry based technologies applied toward the study of proteins. Equally notable is the rapid adaptation of these technologies to biomedical approaches in the realm of clinical proteomics. Concerted efforts toward the elucidation of the proteomes of organ sites or specific disease state are proliferating and from these efforts come the promise of better diagnostics/prognostics and therapeutic intervention. Prostate cancer has been a focus of many such studies with the promise of improved care to patients via biomarkers derived from these proteomic approaches. The newer technologies provide higher analytical capabilities, employ automated liquid handling systems, fractionation techniques and bioinformatics tools for greater sensitivity and resolving power, more robust and higher throughput sample processing, and greater confidence in analytical results. In this prospects, we summarize the proteomic technologies applied to date in prostate cancer, along with their respective advantages and disadvantages. The development of newer proteomic strategies for use in future applications is also discussed.     

USING CARBON AND NITROGEN ISOTOPE VALUES TO INVESTIGATE MATERNAL STRATEGIES IN NORTHEAST PACIFIC OTARIIDS

We examine the utility of stable carbon and nitrogen isotope variations to characterize the length of the nursing/lactation period and age at weaning for two northern Pacific otariid species, the northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus). We used two sampling strategies to measure ontogenetic trends in isotope value, and compared our results to observational data on the reproductive strategies used by these otariids. For Zalophus, we found evidence for ¹⁵N enrichment and ¹³C‐depletion in bone collagen representing the first and second year of growth, which is consistent with the ～12–14‐mo weaning age in this population after a suitable turnover rate for bone collagen is considered. Analysis of individual tooth annuli from a different suite of Zalophus specimens suggests that half of the individuals were weaned at ～12 mo of age, and half were dependent on milk for a portion of their second year. For Callorhinus, bone collagen for age classes that contain pre‐weaned individuals were ¹⁵N‐enriched, but values were significantly lower in specimens between 6 and 20 mo of age. These ¹⁵N‐enriched values, presumably acquired during nursing between 0 and 4 mo of age in Callorhinus, were not present in specimens older than 12 mo of age. Thus complete bone collagen turnover in young‐of‐the‐year occurs in 8–10 mo. ¹⁵N enrichment is evident in the first annulus of female Callorhinus individuals, but is not detectable in males. Analyses of Callorhinus tooth annuli show no ontogenetic trends in δ¹³C values. Our study indicates that nitrogen, and in some cases carbon, isotopes can be used to assess reproductive strategies in marine mammals. When coupled with accurate age estimates based on bone growth regressions, this isotopic technique can be applied to historical or fossil otariids to gain insight into the flexibility of maternal strategies within and across species.     

ADP-ribosylation of dinitrogenase reductase in Azospirillum brasilense is regulated by AmtB-dependent membrane sequestration of DraG

Nitrogen fixation in some diazotrophic bacteria is regulated by mono-ADP-ribosylation of dinitrogenase reductase (NifH) that occurs in response to addition of ammonium to the extracellular medium. This process is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG), but the means by which the activities of these enzymes are regulated are unknown. We have investigated the role of the P(II) proteins (GlnB and GlnZ), the ammonia channel protein AmtB and the cellular localization of DraG in the regulation of the NifH-modification process in Azospirillum brasilense. GlnB, GlnZ and DraG were all membrane-associated after an ammonium shock, and both this membrane sequestration and ADP-ribosylation of NifH were defective in an amtB mutant. We now propose a model in which membrane association of DraG after an ammonium shock creates a physical separation from its cytoplasmic substrate NifH thereby inhibiting ADP-ribosyl-removal. Our observations identify a novel role for an ammonia channel (Amt) protein in the regulation of bacterial nitrogen metabolism by mediating membrane sequestration of a protein other than a P(II) family member. They also suggest a model for control of ADP-ribosylation that is likely to be applicable to all diazotrophs that exhibit such post-translational regulation of nitrogenase.     

Romping the cellular landscape: linear scaffolds for molecular recognition

Multivalent molecules with a precise array of recognition elements that interact with specific cell types are important for characterizing the topology of molecules on a cell surface. Applications ranging from the control of cellular signaling to drug delivery and tissue imaging rely on these surface-mapping molecules. Linear polymers provide a molecular scaffold that is advantageous for these types of applications and their synthesis can be amenable to the introduction of different recognition elements. Recently, advances have been made in the development of synthetic approaches for preparing linear polymeric substrates with highly controlled lengths and recognition element spacing.     

Potent hFPRL1 (ALXR) agonists as potential anti-inflammatory agents

We report the discovery of potent agonists for the human formyl-peptide-like 1 receptor (hFPRL1). These compounds did not act at a closely related receptor denoted human formyl peptide receptor (hFPR) up to 10 microM concentration. Recent studies have indicated that agonizing this receptor may promote resolution of inflammation. In an exploratory study, a novel hFPRL1 agonist showed efficacy in a mouse ear inflammation model following oral administration.