Translocation efficiency of apolipoprotein B is determined by the presence of beta-sheet domains, not pause transfer sequences

Cotranslational translocation of apoB100 across the endoplasmic reticulum (ER) membrane is inefficient, resulting in exposure of nascent apoB on the cytosolic surface of the ER. This predisposes apoB100 to ubiquitinylation and targeting for proteasomal degradation. It has been suggested that pause transfer sequences (PTS) present throughout apoB cause inefficient translocation. On the other hand, our previous study demonstrated that the translocation efficiency of apoB100 is dependent on the presence of a beta-sheet domain between 29 and 34% of full-length apoB100 (Liang, J.-S., Wu, X., Jiang, H., Zhou, M., Yang, H., Angkeow, P., Huang, L.-S., Sturley, S. L., and Ginsberg, H. N. (1998) J. Biol. Chem. 273, 35216-35221); this region of apoB has no PTS. However, the effects of the beta-sheet domain may require the presence of PTS elsewhere in the N-terminal region of apoB100. To further investigate the roles of PTS and beta-sheet domains in the translocation of apoB100 across the ER, we transfected McArdle RH7777, HepG2, or Chinese hamster ovary cells with human albumin (ALB)/human apoB chimeric cDNA constructs: ALB/B12-17 (two PTS but no beta-sheet), ALB/B29-34 (beta-sheet but no PTS), ALB/B36-41 (two PTS and a beta-sheet), and ALB/B49-54 (neither PTS nor a beta-sheet). ALB/ALB1-40 served as a control. Compared with ALB/ALB1-40, secretion rates of ALB/B12-17, ALB/B29-34, and ALB/B36-41 were reduced. Secretion of ALB/B49-54 was similar to that of ALB/ALB1-40. However, only ALB/B29-34 and ALB/B36-41 had increased proteinase K sensitivity, ubiquitinylation, and increased physical interaction with Sec61alpha. These results indicate that the translocation efficiency of apoB100 is determined mainly by the presence of beta-sheet domains. PTS do not appear to affect translocation, but may affect secretion by other mechanisms.     

Antimicrobial peptides from diverse families isolated from the skin of the Asian frog, Rana grahami

Seven peptides with antimicrobial activity were isolated in pure form from an extract of the skin of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. The peptides were identified as belonging to the nigrocin-2 (three peptides), brevinin-1 (one peptide), brevinin-2 (three peptides), and esculentin-1 (one peptide) families. Nigrocin-2GRb (GLFGKILGVGKKVLCGLSGMC) containing three lysine residues, represented the peptide with highest potency against microorganisms (MIC = 3 microM against Escherichia coli, 12.5 microM against Staphylococcus aureus and 50 microM against Candida albicans) and the greatest hemolytic activity against human erythrocytes (LD50 = 40 microM). In contrast, nigrocin-2GRa (GLLSGILGAGKHIVCGLSGLC) and nigrocin-2GRc (GLLSGILGAGKNIVCGLSGLC), with only a single lysine residue, showed weak antimicrobial and hemolytic activity. Phylogenetic relationships among Eurasian ranid frogs are less well understood than those of North American ranids but the primary structures of the R. grahami antimicrobial peptides suggest a close relationship of this species with the Japanese pond frogs R. nigromaculata and R. porosa brevipoda.     

TBX5 is required for embryonic cardiac cell cycle progression

Despite the critical importance of TBX5 in normal development and disease, relatively little is known about the mechanisms by which TBX5 functions in the embryonic heart. Our present studies demonstrate that TBX5 is necessary to control the length of the embryonic cardiac cell cycle, with depletion of TBX5 leading to cardiac cell cycle arrest in late G(1)- or early S-phase. Blocking cell cycle progression by TBX5 depletion leads to a decrease in cardiac cell number, an alteration in the timing of the cardiac differentiation program, defects in cardiac sarcomere formation, and ultimately, to cardiac programmed cell death. In these studies we have also established that terminally differentiated cardiomyocytes retain the capacity to undergo cell division. We further show that TBX5 is sufficient to determine the length of the embryonic cardiac cell cycle and the timing of the cardiac differentiation program. Thus, these studies establish a role for TBX5 in regulating the progression of the cardiac cell cycle.     

Norbornyllactone-substituted xanthines as adenosine A(1) receptor antagonists

During the search for second-generation adenosine A(1) receptor antagonist alternatives to the clinical candidate 8-(3-oxa-tricyclo[3.2.1.0(2,4)]oct-6-yl)-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (BG9719), we developed a series of novel xanthines substituted with norbornyl-lactones that possessed high binding affinities for adenosine A(1) receptors and in vivo activity.     

Development of potent anti-infective agents from Silurana tropicalis: Conformational analysis of the amphipathic,alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7

Peptide XT-7 (GLLGP⁵LLKIA¹⁰AKVGS¹⁵NLL.NH₂) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC₅₀ = 140 µM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC₅₀ > 500 µM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d₃)-H₂O mixed solvent system, XT-7 is characterised by a right handed α-helical conformation between residues Leu³ and Leu¹⁷ whereas [G4K]XT-7 adopts a more restricted α-helical conformation between residues Leu⁶ and Leu¹⁷. A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu³ and Leu¹⁷. However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H₂O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly⁴ → Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.     

Focal adhesion kinase is essential for cardiac looping and multichamber heart formation

Focal adhesion kinase (FAK) is a critical mediator of matrix‐ and growth factor‐induced signaling during development. Myocyte‐restricted FAK deletion in mid‐gestation mice results in impaired ventricular septation and cardiac compaction. However, whether FAK regulates early cardiogenic steps remains unknown. To explore a role for FAK in multi‐chambered heart formation, we utilized anti‐sense morpholinos to deplete FAK in Xenopus laevis. Xenopus FAK morphants exhibited impaired cardiogenesis, pronounced pericardial edema, and lethality by tadpole stages. Spatial‐temporal assessment of cardiac marker gene expression revealed that FAK was not necessary for midline migration, differentiation, fusion of cardiac precursors, or linear heart tube formation. However, myocyte proliferation was significantly reduced in FAK morphant heart tubes and these tubes failed to undergo proper looping morphogenesis. Collectively our data imply that FAK plays an essential role in chamber outgrowth and looping morphogenesis likely stimulated by fibroblast growth factors (and possibly other) cardiotrophic factors. genesis 48:492–504, 2010. © 2010 Wiley‐Liss, Inc.     

Smoking (active and passive), N-acetyltransferase 2, and risk of breast cancer

Background: The relationship between smoking and breast cancer remains controversial. The study aim was to assess the relationship of passive and active smoking to breast cancer risk by N-acetyltransferase 2 (NAT2) phenotype, using a comprehensive assessment of both passive and active smoking. Methods: We undertook a population-based case–control study in Northeastern Ontario, Canada of 347 women diagnosed (2002–2004) with breast cancer and 775 population-based controls. The mailed study package included a questionnaire requesting information about established breast cancer risk factors, passive and active smoking, and a buccal swab for genetic analyses. Results: Among never-active smokers, a long duration of passive smoking was associated with an increased risk of breast cancer (odds ratio (OR) 1.86 (95% confidence interval (95% CI) 1.01–3.44) (test for trend (p = 0.07)); that risk was more elevated for NAT2 slow acetylators (OR 2.76, 95% CI 1.16–6.59) (and highest in extremely slow acetylators), but not elevated for NAT2 fast acetylators (OR 1.17, 95% CI 0.42–3.23). Among active smokers more than 20 pack-years of smoking was associated with an OR of 1.34 (95% CI 0.92-1.96); more elevated among NAT2 fast acetylators OR 1.93 (95% CI 1.01–3.69) but not elevated among NAT2 slow acetylators. Women who were NAT2 fast acetylators in the highest quartile for duration of active smoking had an OR of 2.74 (95% CI 1.42–5.27), with a significant test of trend (p = 0.005). Conclusions: These findings suggest that passive and active smoking may be related to breast cancer, and the effect may be differentially modified by NAT2 phenotype. Further research into the genetic modification of a breast cancer–smoking relationship may help to reconcile earlier discrepant findings.     

The primary structures and myotropic activities of two tachykinins isolated from the African clawed frog,Xenopus laevis

Two peptides with limited structural similarity to mammalian substance P (SP) and neurokinin A (NKA) have been isolated from extracts of the intestine of the African clawed frog (Xenopus laevis). The primary structure of an SP-like peptide was established as: Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met.NH(2), which is identical to the previously characterized peptide, bufokinin isolated from the toad Bufo marinus. The primary structure of an NKA-related peptide was established as Thr-Leu-Thr-Thr-Gly-Lys-Asp-Phe-Val-Gly-Leu-Met.NH(2). Only the five amino acids at the C-terminal region of the peptide are identical to mammalian NKA whereas the N-terminal region shows no structural similarity to previously characterized tachykinins. Immunohistochemical investigations of the gut wall revealed a dense network of nerve fibres and nerve cell bodies containing SP/NKA-like substances. The myotropic effects of the Xenopus tachykinins were compared with the contractile effect of mammalian SP and NKA on isolated strips of circular smooth muscle from Xenopus stomach. No significant differences in potencies (-log EC(50)) or in intrinsic activities were observed between the Xenopus and mammalian peptides. The potencies for the Xenopus SP-like (8.49+/-0.15) and the NKA-like peptide (8.12+/-0.06) were similar suggesting that the amino acid sequence at the N-terminal region of the tachykinins is not important in activating the tachykinin receptors in Xenopus gastric smooth muscle. The maximum response to Xenopus SP (alpha=0.59+/-0.06) was significantly lower than to the NKA-like peptide (alpha=1.0) suggesting a more effective interaction of the NKA-like peptide with the tachykinin receptor(s) in Xenopus stomach.     

The temperature dependence of erythrocyte water diffusion permeability

1. The activation energy of the diffusion water exchange in red blood cells increases with temperature. 2. Fetal blood has a higher activation energy for diffusion water exchange than adult blood. 3. Treatment of red cells with p-chloromercuribenzoate alters the activation energy and apparently allows a lipid and a protein pore pathway to be resolved. The permeability and activation energy of the treated cells is in the range found for lipid membranes; and the difference between treated and untreated cells, the "protein" pathway, has a diffusion activation energy comparable with that of free water. 4. A resolution of the discrepancies between the NMR methods of measuring diffusion water exchange is suggested.