Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.     

Cdon Mutation and Fetal Ethanol Exposure Synergize to Produce Midline Signaling Defects and Holoprosencephaly Spectrum Disorders in Mice

Holoprosencephaly (HPE) is a remarkably common congenital anomaly characterized by failure to define the midline of the forebrain and midface. HPE is associated with heterozygous mutations in Sonic hedgehog (SHH) pathway components, but clinical presentation is extremely variable and many mutation carriers are unaffected. It has been proposed that these observations are best explained by a multiple-hit model, in which the penetrance and expressivity of an HPE mutation is enhanced by a second mutation or the presence of cooperating, but otherwise silent, modifier genes. Non-genetic risk factors are also implicated in HPE, and gene–environment interactions may provide an alternative multiple-hit model to purely genetic multiple-hit models; however, there is little evidence for this contention. We report here a mouse model in which there is dramatic synergy between mutation of a bona fide HPE gene (Cdon, which encodes a SHH co-receptor) and a suspected HPE teratogen, ethanol. Loss of Cdon and in utero ethanol exposure in 129S6 mice give little or no phenotype individually, but together produce defects in early midline patterning, inhibition of SHH signaling in the developing forebrain, and a broad spectrum of HPE phenotypes. Our findings argue that ethanol is indeed a risk factor for HPE, but genetically predisposed individuals, such as those with SHH pathway mutations, may be particularly susceptible. Furthermore, gene–environment interactions are likely to be important in the multifactorial etiology of HPE.     

Penicillin-binding protein 2x of Streptococcus pneumoniae: three new mutational pathways for remodelling an essential enzyme into a resistance determinant

Mutations in the transpeptidase domain of penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae that reduce the affinity to beta-lactams are important determinants of resistance to these antibiotics. We have now analyzed in vitro and in vivo properties of PBP2x variants from cefotaxime-resistant laboratory mutants and a clinical isolate. The patterns of two to four resistance-specific mutations present in each of the proteins, all of which are placed between 6.6 and 24 Å around the active site, fall into three categories according to their positions in the three-dimensional structure. The first PBP2x group is characterized by mutations at the end of helix α11 and carries the well-known T550A change and/or one mutation on the surface of the penicillin-binding domain in close contact with the C-terminal domain. All group I proteins display very low acylation efficiencies, ≤ 1700 M^− 1 s^− 1, for cefotaxime. The second class represented by PBP2x of the mutant C505 shows acylation efficiencies below 100 M^− 1 s^− 1 for both cefotaxime and benzylpenicillin and contains the mutation L403F at a critical site close to the active serine. PBP2x of the clinical isolate 669 reveals a third mutational pathway where at least the two mutations Q552E and S389L are important for resistance, and acylation efficiency is reduced for both beta-lactams to around 10,000 M^− 1 s^− 1. In each group, at least one mutation is located in close vicinity to the active site and mediates a resistance phenotype in vivo alone, whereas other mutations might exhibit secondary effects only in context with other alterations.     

Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase

Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.     

Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria

Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.     

Polymorphic microsatellite DNA markers for Banksia hookeriana (Proteaceae)

We developed 11 polymorphic microsatellite DNA markers for an Australian native shrub Banksia hookeriana (Proteaceae). The number of alleles per locus in 37 individuals varied from three to 17, observed and expected heterozygosities ranged from 0.297 to 0.838 and from 0.279 to 0.900, respectively. Two loci (BH‐B5 and BH‐B107) showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05), and null alleles may be present at these two loci. All loci showed independent inheritance.     

Probing the functional tolerance of the b subunit of Escherichia coli ATP synthase for sequence manipulation through a chimera approach

A dimer of 156-residue b subunits forms the peripheral stator stalk of eubacterial ATP synthase. Dimerization is mediated by a sequence with an unusual 11-residue (hendecad) repeat pattern, implying a right-handed coiled coil structure. We investigated the potential for producing functional chimeras in the b subunit of Escherichia coli ATP synthase by replacing parts of its sequence with corresponding regions of the b subunits from other eubacteria, sequences from other polypeptides having similar hendecad patterns, and sequences forming left-handed coiled coils. Replacement of positions 55-110 with corresponding sequences from Bacillus subtilis and Thermotoga maritima b subunits resulted in fully functional chimeras, judged by support of growth on nonfermentable carbon sources. Extension of the T. maritima sequence N-terminally to position 37 or C-terminally to position 124 resulted in slower but significant growth, indicating retention of some capacity for oxidative phosphorylation. Portions of the dimerization domain between 55 and 95 could be functionally replaced by segments from two other proteins having a hendecad pattern, the distantly related E subunit of the Chlamydia pneumoniae V-type ATPase and the unrelated Ag84 protein of Mycobacterium tuberculosis. Extension of such sequences to position 110 resulted in loss of function. None of the chimeras that incorporated the leucine zipper of yeast GCN4, or other left-handed coiled coils, supported oxidative phosphorylation, but substantial ATP-dependent proton pumping was observed in membrane vesicles prepared from cells expressing such chimeras. Characterization of chimeric soluble b polypeptides in vitro showed their retention of a predominantly helical structure. The T. maritima b subunit chimera melted cooperatively with a midpoint more than 20 degrees C higher than the normal E. coli sequence. The GCN4 construct melted at a similarly high temperature, but with much reduced cooperativity, suggesting a degree of structural disruption. These studies provide insight into the structural and sequential requirements for stator stalk function.     

Environmental drivers in mangrove establishment and early development: A review

Mangroves have a global distribution within coastal tropical and subtropical climates, and have even expanded to some temperate locales. Where they do occur, mangroves provide a plethora of goods and services, ranging from coastal protection from storms and erosion to direct income for human societies. The mangrove literature has become rather voluminous, prompting many subdisciplines within a field that earlier in the 20th century received little focus. Much of this research has become diffuse by sheer numbers, requiring detailed syntheses to make research results widely available to resource managers. In this review, we take an inclusive approach in focusing on eco-physiological and growth constraints to the establishment and early development of mangrove seedlings in the intertidal zone. This is a critical life stage for mangroves, i.e., the period between dispersal and recruitment to the sapling stage. We begin with some of the research that has set the precedent for seedling-level eco-physiological research in mangroves, and then we focus on recent advances (circa. 1995 to present) in our understanding of temperature, carbon dioxide, salinity, light, nutrient, flooding, and specific biotic influences on seedling survival and growth. As such, we take a new approach in describing seedling response to global factors (e.g., temperature) along with site-specific factors (e.g., salinity). All variables will strongly influence the future of seedling dynamics in ways perhaps not yet documented in mature forests. Furthermore, understanding how different mangrove species can respond to global factors and regional influences is useful for diagnosing observed mortality within mangrove wetlands, managed or natural. This review provides an updated eco-physiological knowledge base for future research and reforestation activity, and for understanding important links among climate change, local physico-chemical condition, and establishment and early growth of mangrove seedlings.     

Potential of aquatic fungi derived from diverse freshwater environments to decolourise synthetic azo and anthraquinone dyes

A total of 37 strains of aquatic hyphomycetes and 95 fungal isolates derived from diverse freshwater environments were screened on agar plates for the decolourisation of the disazo dye Reactive Black 5 and the anthraquinone dye Reactive Blue 19. The decolourisation of 9 azo and 3 anthraquinone dyes by 9 selected aquatic fungi was subsequently assessed in a liquid test system. The fungi were representatives of mitosporic anamorphs, and 6 strains had proven ascomycete affiliations. For comparison, 5 white rot basidiomycetes were included. The majority of dyes were decolourised by several mitosporic aquatic isolates at rates essentially comparable to those observed with the most efficient white rot fungus. Under certain conditions, particular aquatic strains decolourised dyes even more efficiently than the best performing white rot basidiomycete. Upon fungal treatment of several dyes, new absorbance peaks appeared, indicating biotransformation metabolites. All together, these results point to the potential of fungi occurring in freshwater environments for the treatment of dye-containing effluents.