Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles

The adsorption of colipase is essential for pancreatic triglyceride lipase activity and efficient dietary fat digestion. Yet, little is known about which specific amino acids in the hydrophobic surface of colipase influence adsorption. In this study, we systematically substituted alanine or tryptophan at residues implicated in adsorption of colipase to an interface. We expressed, purified recombinant colipase mutants and characterized the ability of each alanine mutant to restore activity to lipase in the presence of bile salts. The functions of L16A, Y55A, I79A and F84A colipase were most impaired with activities ranging from 20 to 60% of wild-type colipase. We next characterized the fluorescence properties of the tryptophan mutants in the absence and presence of bile–salt–oleic acid mixed micelles. We performed steady-state emission spectra to determine peak shift and I₃₃₀/I₃₅₀ ratio and acrylamide quenching curves to characterize the environment of the residues. The analysis supports a model of adsorption that includes residues Leu 34 and Leu 36 on the 2nd loop, Tyr 55 and Tyr 59 on the 3rd loop and Ile 75 and Ile 79 on the 4th loop. The analysis confirms that Phe 84 is not part of the adsorption surface and likely stabilizes the conformation of colipase. Contrary to the predictions of computer modeling, the results provide strong support for an essential role of Tyr 55 in colipase adsorption to mixed micelles. The results indicate that the adsorption of colipase to mixed micelles is mediated by specific residues residing in a defined surface of colipase.     

Lifestyle impacts on the aging‐associated expression of biomarkers of DNA damage and telomere dysfunction in human blood

Cellular aging is characterized by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and activation of DNA damage responses. There is some evidence that DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage, and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging‐associated expression of serum markers of DNA damage (CRAMP, EF‐1α, stathmin, n‐acetyl‐glucosaminidase and chitinase) in comparison with other described markers of cellular aging (p16^INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age‐independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age‐independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16^INK4a expression and negatively with telomere length in peripheral blood T‐lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging.     

Molecular steps of G‐overhang generation at human telomeres and its function in chromosome end protection

Telomeric G‐overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G‐overhang generation at human telomeres is poorly understood. Here, we show that G‐overhangs can undergo cell cycle‐regulated changes independent of telomerase activity. G‐overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C‐strand fill‐in, whereas the sizes of G‐overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C‐strands are precisely defined throughout the cell cycle, indicating that C‐strand resection is strictly regulated. We demonstrate that C‐strand fill‐in is mediated by DNA polymerase α (polα) and controlled by cyclin‐dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G‐overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G‐overhangs and an increase in telomeric DNA damage. Our results suggest that G‐overhang generation at human telomeres is regulated by multiple tightly controlled processes and C‐strand fill‐in is under the control of polα and CDK1.     

Identification and characterization of a nuclear factor that specifically binds to the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1)

The Rev protein of human immunodeficiency virus type 1 (HIV-1) differentially transactivates the expression of viral structural proteins by allowing the accumulation of unspliced and singly spliced viral mRNA in the cytoplasm. The cis-acting RNA target sequence for the Rev protein, termed the Rev response element (RRE), is present in the env gene and is predicted to form a highly ordered RNA secondary structure. Recent data indicate that Rev directly binds to RRE and, further, that this binding can be mapped to a 90-nucleotide subfragment at the 5' end of RRE. We now report that RRE also binds specifically and predominantly to a nuclear factor of approximately 56 kD. Mapping of the binding site reveals that the same subfragment that binds Rev also binds this nuclear factor. We designate this protein as NFRRE for nuclear factor, RRE binding. Rev and NFRRE appear to bind simultaneously to RRE. NFRRE is widely distributed in various mammalian cells. We speculate that this factor plays an important role in Rev-mediated transactivation and is likely to be involved in the processing or transport of cellular mRNA.     

Expression of a functional recombinant C‐type lectin‐like protein lebecetin in the human embryonic kidney cells

Lebecetin is an anticoagulant C‐type lectin‐like protein that was previously isolated from Macrovipera lebetina venom and described to consist of two subunits (alpha and beta). It was reported to potently prevent platelet aggregation by binding to glycoprotein Ib and to exhibit a broad spectrum of inhibitory activities on various integrin‐mediated functions of tumor cells, including adhesion, proliferation, and cell migration. This study aimed to investigate the structure‐function of lebecetin. Accordingly, the cDNA of each subunit was cloned and separately or jointly expressed in the human embryonic kidney cells using two vectors with different selectable tags. The immunofluorescence analysis of transfected cells revealed significant expression levels and co‐localization of the two lebecetin subunits. The recombinant proteins were efficiently secreted and purified using metal‐chelating affinity chromatography. We found that the Lebecetin alpha and beta subunits were produced as a mixture of homodimers and heterodimers and that the heterodimerization represents a prerequisite for functioning. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Identification of a cis-acting element in human immunodeficiency virus type 2 (HIV-2) that is responsive to the HIV-1 rev and human T-cell leukemia virus types I and II rex proteins.

A simian virus 40 late replacement vector encoding human immunodeficiency virus type 1 (HIV-1) gp120 (pGP120) was used to define a region within the HIV-2 genome that could work as a rev-responsive element (RRE). Our previous work showed that gp120 expression in this system required a functional RRE in cis and required the rev protein in trans (M.-L. Hammarskj?ld, J. Heimer, B. Hammarskj?ld, I. Sangwan, L. Albert, and D. Rekosh, J. Virol. 63:1959-1966, 1989). Using pGP120, we first mapped an RRE to a 1,042-base-pair (bp) Sau3a fragment in the env region of HIV-2. Both HIV-1 rev (rev1) and HIV-2 rev (rev2) could work in conjunction with this fragment. Further mapping showed that a 272-bp subfragment within the 1,042-bp region was sufficient as an RRE. Surprisingly, the smaller fragment worked only with the rev1 protein and not with its homologous rev2 protein. In addition, the rev2 protein failed to function together with the RRE from HIV-1. We also utilized this system to examine the ability of the rex genes of human T-cell leukemia virus types I and II to functionally substitute for rev. These experiments showed that complementation by both the rexI and rexII proteins required the presence of an RRE. The rex proteins worked well in conjunction with either the HIV-1 or the HIV-2 RRE (the 1,042-bp as well as the 272-bp fragment).     

Trophic positioning and microphytobenthic carbon uptake of biofilm‐dwelling meiofauna in a temperate river

1. δ¹³C and δ¹⁵N stable isotope signatures combined with an in situ microphytobenthic ¹³C labelling experiment were performed on epilithic biofilms of a large temperate river (the Garonne, France) to infer the trophic positioning of biofilm‐dwelling meiofauna and their uptake of microphytobenthic carbon. 2. Chironomidae larvae and Chromadorina spp. nematodes rapidly incorporated freshly produced microphytobenthic carbon in contrast to Rhyacophilidae larvae and Naididae oligochaetes. Quantitatively, macrofaunal Chironomidae incorporated more microphytobenthic carbon per day than did meiofauna. Moreover, Chironomidae seemed more involved in the spatial export of microphytobenthic carbon than nematodes. 3. Rhyacophilidae larvae were predators feeding on large meiofauna (Naididae and Chironomidae) but not on nematodes. Naididae oligochaetes primarily gained their carbon from allochthonous and/or microbial‐loop recycled sources. 4. A rapid and significant loss of labelled microphytobenthic carbon was observed. Feeding activity of biofilm‐dwelling invertebrates seemed not to be primarily involved in this loss.     

Benzo[a]pyrene-induced cell cycle progression is through ERKs/cyclin D1 pathway and requires the activation of JNKs and p38 mapk in human diploid lung fibroblasts

Treatment of cells with carcinogen Benzo[a]pyrene (B[a]P) allows cells to evade G1 arrest and induces cells abnormal proliferation. However, the mechanisms of its action at cellular level are not well understood. To address this question, normal human embryo lung diploid fibroblasts (HELF) were selected in the present study. We found that exposure of cells with 2.5 μM of B[a]P for 24 h resulted in a decrease of G1 population by 11.9% (P < 0.05) and a increase of S population by 17.2% (P < 0.05). Treatment of cells with B[a]P also caused dose-related activation of MAPK and induction of cyclin D1 protein expression, whereas the CDK4 protein levels were not significantly affected by B[a]P. Overexpression of cyclin D1 protein stimulated by B[a]P was significantly inhibited by 50 μM AG126 (an inhibitor of ERK1/2), but not by 25 μM SP600125 (an inhibitor of JNK1/2) or 5 μM SB203580 (an inhibitor of p38 mapk), suggesting that B[a]P-induced cyclin D1 expression was only regulated by ERK1/2 pathway. However, AG126, SP600125 or SB203580 led to cell cycle significantly arrested in G1 phase, indicating that ERK1/2, JNK1/2 and p38 mapk pathways are all required for B[a]P-induced G1/S transition. In addition, HELF cells transfecting with antisense cyclin D1 cDNA or antisense CDK4 cDNA showed significantly G1 arrest after B[a]P stimulation. These results suggested that B[a]P exposure accelerated the G1→S transition by activation of MAPK signaling pathways. Cyclin D1 and CDK4 are rate-limiting regulators of the G1→S transition and expression of cyclin D1 is predominantly regulated by ERK1/2 pathway in HELF cells.     

Identification of caspase 3 motifs and critical aspartate residues in human phospholipase D1b and phospholipase D2a

Stimulation of mammalian cells frequently initiates phospholipase D-catalyzed hydrolysis of phosphatidylcholine in the plasma membrane to yield phosphatidic acid (PA) a novel lipid messenger. PA plays a regulatory role in important cellular processes such as secretion, cellular shape change, and movement. A number of studies have highlighted that PLD-based signaling also plays a pro-mitogenic and pro-survival role in cells and therefore anti-apoptotic. We show that human PLD1b and PLD2a contain functional caspase 3 cleavage sites and identify the critical aspartate residues within PLD1b that affect its activation by phorbol esters and attenuate phosphatidylcholine hydrolysis during apoptosis.     

Identification of a new adhesin‐like protein from Lactobacillus mucosae ME‐340 with specific affinity to the human blood group A and B antigens

Aims: To identify and characterize a new adhesin‐like protein of probiotics that show specific adhesion to human blood group A and B antigens. Methods and Results: Using the BIACORE assay, the adhesion of cell surface components obtained from four lactobacilli strains that adhered to blood group A and B antigens was tested. Their components showed a significant adhesion to A and B antigens when compared to the bovine serum albumin (BSA) control. The 1 mol l^?1 GHCl fraction extracted from Lactobacillus mucosae ME‐340 contained a 29‐kDa band (Lam29) using SDS–PAGE. The N‐terminal amino acid sequence and homology analysis showed that Lam29 was 90% similar to the substrate‐binding protein of the ATP‐binding cassette (ABC) transporter from Lactobacillus fermentum IFO 3956. The complete nucleotide sequence (858 bp) of Lam29 was determined and encoded a protein of 285 amino acid residues. Phylogenetic analysis and multiple sequence alignments indicated this protein may be related to the cysteine‐binding transporter. Conclusions: The adhesion of ME‐340 strain to blood group A and B antigens was mediated by Lam29 that is a putative component of ABC transporter as an adhesin‐like protein. Significance and Impact of the Study: Lactobacillus mucosae ME‐340 expressing Lam29 may be useful for competitive exclusion of pathogens via blood group antigen receptors in the human gastrointestinal mucosa and in the development of new probiotic foods.     

Genetic alteration of UDP‐rhamnose metabolism in Botrytis cinerea leads to the accumulation of UDP‐KDG that adversely affects development and pathogenicity

Botrytis cinerea is a model plant‐pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)‐glucose, the major fungal wall nucleotide sugar precursor, to UDP‐rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose‐containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP‐rhamnose pathway intermediate UDP‐4‐keto‐6‐deoxy‐glucose (UDP‐KDG) in hyphae of the Δbcer strain. UDP‐KDG could not be detected in hyphae of the wild‐type strain, indicating fast conversion to UDP‐rhamnose by the BcEr enzyme. The correlation between high UDP‐KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP‐KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP‐KDG may lead to the development of new antifungal drugs.     

Identification of a novel point mutation in ENT1 that confers resistance to Ara-C in human T cell leukemia CCRF-CEM cells

The genetic basis for the Ara-C resistance of CCRF-CEM Ara-C/8C leukemia cells was investigated. DNA sequencing revealed that these cells expressed an equilibrative nucleoside transporter 1 (ENT1) with a single missense mutation resulting in glycine to arginine replacement (G24R). To test the importance of this residue, additional G24 mutants were created and examined for [3H]-uridine and [3H]-Ara-C uptake. Both a G24E and G24A mutant showed reduced ENT1-dependent activity. An EGFP-tagged G24R ENT1 displayed plasma membrane localization even though it was unable to bind [3H]-NBMPR, an ENT1-specific inhibitor. These results define G24 as critical amino acid for ENT1 nucleoside uptake and suggest that mutations in TM1 may provide a mechanism for Ara-C resistance in CCRF-CEM Ara-C/8C cells.