HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF PHYTOPLANKTON PIGMENTS USING A POLYMERIC REVERSED-PHASE C18 COLUMN1

A high-performance liquid chromatographic (HPLC) method is described that allows improved resolution of several chemotaxonomically significant phytoplankton pigments. The protocol, which employs two pumps and a modified Mantoura and Llewellyn (1983) solvent system, can be easily adapted for many HPLC systems currently in use. The most unique aspect of the method is the use of a polymeric C₁₈ reversed phase HPLC column (VYDAC 201TP). In comparison to the monomeric C₁₈ columns typically used in the characterization of phytoplankton pigments, polymeric C₁₈ columns offer superior selectivity for structurally similar compounds. The protocol was evaluated for the ability to resolve most of the phytoplankton pigments of diagnostic importance using algal cultures from nine classes. Pigment pairs that were resolved by the method include a) lutein and zeaxanthin, b) neoxanthin and 19′-hexanoyloxyfucoxanthin, and c) α-carotene and β-carotene, and partial resolution of chlorophyll c₁ and chlorophyll c₂.     

Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.     

11β-Hydroxysteroid dehydrogenase-type 2 evolved from an ancestral 17β-Hydroxysteroid dehydrogenase-type 2

11β-Hydroxysteroid dehydrogenase-type 2 (11β-HSD2) regulates the local concentration of cortisol that can activate the glucocorticoid receptor and mineralocorticoid receptor, as well as the concentration of 11-keto-testosterone, the active androgen in fish. Similarly, 17β-HSD2 regulates the levels of testosterone and estradiol that activate the androgen receptor and estrogen receptor, respectively. Interestingly, although human 11β-HSD2 and 17β-HSD2 act at different positions on different steroids, these enzymes are paralogs. Despite the physiological importance of 11β-HSD2 and 17β-HSD2, details of their origins and divergence from a common ancestor are not known. An opportunity to understand their evolution is presented by the recent sequencing of genomes from sea urchin, a basal deuterostome, and amphioxus, a basal chordate, and the availability of substantial sequence for acorn worm and elephant shark, which together provide a more complete dataset for analysis of the origins of 11β-HSD2 and 17β-HSD2. BLAST searches find an ancestral sequence of 17β-HSD2 in sea urchin, acorn worm and amphioxus, while an ancestral sequence of 11β-HSD2 first appears in sharks. Sequence analyses indicate that 17β-HSD2 in sea urchin may have a non-enzymatic activity. Evolutionary analyses indicate that if acorn worm 17β-HSD2 is catalytically active, then it metabolizes novel substrate(s).     

Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.     

Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis

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Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

Markov modelling of immunological and virological states in HIV-1 infected patients

The purpose of this study was to evaluate the evolution of HIV infected patients and to bring out some significant factors associated with this pathology. The main criteria revealing the State of illness is viral load measurement (VL). However the CD4 lymphocytes also represent an important marker as these reflect the State of the immune reservoir. Many studies have been carried out in this field and different models have been proposed with a view to a better understanding of this disease. Multi State Markov models defined in terms of CD4 counts, or in terms of viral load, have proved to be very useful tools for modelling HIV disease progression. The model we have developed in this study is based on both the CD4 lymphocytes counts and VL. Markov models are characterized by transition intensities. In this paper we explored several structures in succession. First, we used a homogeneous continuous time Markov process with four states defined by crossed values of CD4 and VL in a given patient at a given time. Then, the effect of certain covariates on the infection process was introduced into the model via the transition intensity functions, as with a Cox regression model. Since the hypothesis of homogeneity may be unrealistic in certain cases, we also considered piecewise homogeneous Markov models. Finally, the effects of covariates and time were combined in a piecewise homogeneous model with a covariate. We applied these methods to data from 1313 HIV-infected patients included in the NADIS cohort.     

Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, α-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred α-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from α-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.     

Ultrasonication of insulin-loaded microgel particles produced by internal gelation: Impact on particle's size and insulin bioactivity

Alginate-dextran sulfate (ADS) microgel has been used to protect insulin from gastrointestinal attack and as a carrier to promote insulin permeation through intestinal epithelium. The throughput of ADS submicron particles generation by emulsification/internal gelation is limited by its wide size distribution.