The mechanism of phosphatidylcholine‐induced interference of PAP (248‐286) aggregation

Seminal amyloids are well known for their role in enhancing HIV infection. Among all the amyloidogenic peptides identified in human semen, PAP_248‐286 was found to be the most active and was termed as semen‐derived enhancer of viral infection (SEVI). Although amyloidogenic nature of the peptide is mainly linked with enhancement of the viral infection, the most active physiological conformation of the aggregated peptide remains inconclusive. Lipids are known to modulate aggregation pathway of a variety of proteins and peptides and constitute one of the most abundant biomolecules in human semen. PAP_248‐286 significantly differs from the other known amyloidogenic peptides, including Aβ and IAPP, in terms of critical concentration, surface charge, fibril morphology, and structural transition during aggregation. Hence, in the present study, we aimed to assess the effect of a lipid, 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC), on PAP_248‐286 aggregation and the consequent conformational outcomes. Our initial observation suggested that the presence of the lipid considerably influenced the aggregation of PAP_248‐286. Further, ZDOCK and MD simulation studies of peptide multimerization have suggested that the hydrophobic residues at C‐terminus are crucial for PAP_248‐286 aggregation and are anticipated to be major DOPC‐interacting partners. Therefore, we further assessed the aggregation behaviour of C‐terminal (PAP_273‐286) fragment of PAP_248‐286 and observed that DOPC possesses the ability to interfere with the aggregation behaviour of both the peptides used in the current study. Mechanistically, we propose that the presence of DOPC causes considerable inhibition of the peptide aggregation by interfering with the peptide's disordered state to β‐sheet transition.     

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11β-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC₅₀ values of 1.6 ± 0.1 and 9.9 ± 0.9 nM (mean ± SEM, n = 3–6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K_i values of 0.8 ± 0.04 and 2.2 ± 0.2 nM (n = 3–4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.     

Trade-offs between postcopulatory riding and mate location in the blue milkweed beetle

In the blue milkweed beetle, Chrysochus cobaltinus (Coleoptera:Chrysomelidae), males remain stationary on females' backs forprolonged periods after a single, brief copulation. Lone malesoften attack pairs, and takeovers, in which rival males replacethe resident on the female's back, are common. I used removalexperiments to measure the effect of postcopulatory riding onlatency to remating. Females whose males were removed were morelikely to remate in the next 2 h than females that were allowedto remain paired. When females were removed, males were alsomore likely to remate in the 2-h period following removal thanwere males allowed to continue riding. This indicates that malesdelay remating by their mates at the expense of mating opportunitieswith additional females. The predicted reproductive successof guarding and nonguarding males was calculated using a modelbased upon experimentally derived latencies to remating andsimulated levels of last male paternity. Guarders outperformednonguarders when the proportion of offspring sired by the lastmale (P₂) was 0.4 or greater. Data on survival and lifetimemating success were used to evaluate survival trade-offs ofpostcopulatory riding. Although males sacrificed feeding timeto ride on females' backs, there was a positive relationshipbetween the proportion of time males spent riding and theirlongevity in the patch. These results indicate that postcopulatoryriding has no survival costs and indicate that postcopulatoryriding can be an effective paternity assurance mechanism evenwhen takeovers are common.     

Production of β-damascenone precursors in cell cultures of Vitis labruscana cv Concord grapes

Summary Callus cultures of Vitis labruscana cv. Concord were initiated from both immature fruit and petiole tissue. Analysis revealed that the acid hydrolyzable precursors to -damascenone, a compound important to the flavor of most grape species, were present in both callus types. Light and increased sucrose concentration were shown to raise the levels of the precursor, some of which are glycosides, in the petiole callus but not in the fruit callus. Addition of -carotene, a biogenic precursor to -damascenone and its precursors and the addition of amitrole, an inhibitor of carotenoid cyclization and production, had no effect upon precursor levels in either tissue. Aroma compounds specific to V. labruscana grapes and other American species of grapes were not found in the callus tissue. Cell suspensions were initiated from the fruit callus and analyzed for the American character compounds as well as -damascenone and its acid hydrolyzable precursors. Once again, no aroma compounds specific to American species were found while -damascenone precursors were present at levels lower than were found in callus on solid media and only during stationary phase of growth.     

Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation

We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.     

Discovery and lead optimisation of a potent,selective and orally bioavailable RARβ agonist for the potential treatment of nerve injury

Oxadiazole replacement of an amide linkage in an RARα agonist template 1, followed by lead optimisation, has produced a highly potent and selective RARβ agonist 4-(5-(4,7-dimethylbenzofuran-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (10) with good oral bioavailability in the rat and dog. This molecule increases neurite outgrowth in vitro and induces sensory axon regrowth in vivo in a rodent model of avulsion and crush injury, and thus has the potential for the treatment of nerve injury.     

Differential chemical diagnosis of primary hyperoxaluria type II. Highly sensitive analysis of optical isomers of glyceric acid by GC/MS as diastereoisomeric derivatives

We established a separation method for the optical isomers of glyceric acid in urine by modifying the derivatization steps of the procedure used for the screening and diagnosis. The trimethylsilyl derivatization step in the mass screening procedure was replaced by O-acetyl-(+)-2-butylation, and the samples were analyzed under equivalent GC/MS conditions by capillary gas chromatography on a DB-5MS column. This method can be applied to cases that show a high urinary concentration of glyceric acid to obtain a differential diagnosis of primary hyperoxaluria type II and d-glyceric aciduria easily. l-Glyceric acid was also isolated from the urine of healthy controls as one of the main peaks.     

Recent advances in the design of RAR α and RAR β agonists as orally bioavailable drugs. A review

Retinoic acid receptors (RARs) α, β, and γ are members of the nuclear receptor superfamily. Compounds which bind to and activate the RARs are termed retinoids which regulate a wide variety of biological processes such as vertebrate embryonic morphogenesis and organogenesis, cell growth arrest, differentiation, and apoptosis, as well as their disorders. Although many synthetic selective RARα, RARβ, and RARγ agonists have been designed and prepared, these have generally been lipophilic acids without good drug-like properties and with low oral bioavailability. Recently this has been changing and drug design approaches to highly potent and selective RARα and RARβ agonists with low lipophilicity that are orally bioavailable and less toxic have been developed, that have a range of potential therapeutic uses. This review covers these new advances.     

Upper limit for DNA packaging by Bacillus subtilis bacteriophage phi 105: isolation of phage deletion mutants by induction of oversized prophages

Summary We have determined the upper size limit for DNA packaging in Bacillus subtilis bacteriophage 105 by examining the plaque-forming and transducing capabilities of lysates made from strains containing prophages of various sizes. The upper size limit for efficient packaging of the phage genome appears to be about 40.2 kb, which is about 1 kb larger than the wild-type genome. This places an upper limit of about 5 kb on the size of insertions that can be accommodated in 105 transfection cloning vectors, such as 105J27. Induction of prophages that exceed the upper limit, followed by selection for plaque formation or transduction, provides a powerful means of isolating phage deletion mutants. A comparison of the location of each deletion with the resultant phenotype has enabled us to identify non-essential regions of the phage genome, and regions that are required for tail biosynthesis and for host cell lysis.     

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 × 10⁶ viable cells ml⁻¹. At the end of the growth period glucose was completely depleted from the culture medium, but l-lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.