31P NMR studies of unsonicated aqueous dispersions of neutral and acidic phospholipids. Effects of phase transitions, p2H and divalent cations on the motion in the phosphate region of the polar headgroup

1. The 129 MHz (non-proton decoupled) and 36.4 MHz (proton decoupled) 31P NMR spectra arising from unsonicated aqueous dispersions of well defined species of phospholipid have been investigated. The phospholipids employed (and the parameters varied) include phosphatidylcholine (temperature), phosphatidylethanolamine (temperature), phosphatidic acid (temperature and p2H) and phosphatidylglycerol (temperature, p2H and Ca2+ (or Mg2+)) concentration. 2. At p2H = 7 the 31 P MNR spectra arising from saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol become progressively broader as the temperature is reduced below the phase transition, demonstrating reduced motion in the phosphate region of the polar headgroup. 3. In the liquid crystalline state at p2H = 7 the molecular dipolar order parameters obtained for saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol and very similar, and are independent of the acyl chain length for species derived from lauric and myristic acid. Thus the motion in the methylene-phosphate-methylene region is similar for these different liquid crystaline phospholipid species. 4. The 31 P NMR spectra of aqueous dispersions of 14:0/14:0 phosphatidic acid display anomalous temperature and p2H dependences. The effective chemical shift anistropy (delta v CSA EFF) at 5 degrees C varies from 71 ppm at p2H = 8.5 to 38 ppm at p2H = 2.5. Further, the motion in the phosphate region is relatively insensitive to the gel or liquid crystalline nature of the hydrocarbon chains. 5. The addition of 40 mol% Ca2+ (or Mg2+) to saturated species of phosphatidylglycerol causes an increase of approx. 20 degrees C in the hydrocarbon phase transition temperature as indicated by 31 P NMR. Equimolar concentrations of Ca2+ increase the transition temperature by approx. 70 degrees C, and no 31P NMR signal could be observed for the very condensed precipitate formed below this temperature. In the liquid crystalline state the motion in the phosphate region of the polar headgroup is not significantly affected by the presence of Ca+ or Mg2+. 6. The 31P NMR spectra obtained from 18 : 1c/18 : 1c phosphatidylethanolamine are consistent with a phase transition from a lamellar to an hexagonal (HII) phase in the region 10-15 degrees C. 7. The observed narrowing of the 31 P NMR spectra of aqueous dispersions of phospholipids as the temperature is raised toward the hydrocarbon transition temperature is discussed in terms of the "pretransition" observed in calorimetric studies.     

Pegylated peptides. V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo

In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF (1-29)-NH2, [Ala15]-hGRF (1-29)-NH2, [desNH2Tyr1, D-Ala2, Ala15]-hGRF(1-29)-NH2 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent. PEG moieties of 750, 2000, 5000 or 10,000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C-terminally pegylated hGRF analogs retained high growth hormone (GH)-releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desNH2Tyr1, D-Ala2, Ala15]-hGRF (1-29)-Gly-Gly-Cys(NH2)-S-Nle-PEG5000 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-Gly-Cys(NH2)-S-Nle-PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12-55-fold that of native hGRF). Relative to their non-pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity.     

No evidence for linkage of a novel CA repeat polymorphism for apoptosis antigen 1 (APO-1 or fas) with type I diabetes in a Caucasian population

Abstract DNA typing of four tetrameric repeat loci (HUMVWA, HUMTH0I, D21SII and HPRT) was carried out in a Chinese Han population from Shanghai (East China) and one from Guangzhou (South-East China) using a quadruplex PCR amplification and detection of the fluorescent-labeled alleles on the ALF DNA sequencer. All loci were in accordance with Hardy-Weinberg equilibrium except for D21S11 in the Guangzhou population. A test for population differentiation showed no statistical difference in the allele frequency distribution between the two populations. Comparison of the allele frequency data with other Chinese Han populations from North and South-West China for the STR loci HUMVWA and HUMTH01 revealed heterogeneity between Northern Chinese Han and Southern Chinese Han, which is in accordance with previous studies on the basis of protein markers.     

An ELISA for brown trout (Salmo trutta) vitellogenin and its use in bioassays for environmental estrogens

An enzyme linked immunosorbent assay (ELISA) was developed for the detection of the egg yolk precursor vitellogenin (Vg) in plasma of brown trout (Salmo trutta). Purified Vg from a 17 beta-estradiol-induced trout was used as the competing antigen in the ELISA which is based on polyclonal antibodies. The ELISA's performance was optimized and characterized. The assay's working range was (25-500 ng ml-1), its sensitivity was (10.5 ng ml-1), and it had an intra-assay coefficient of variation of less than 10% between 30 and 1000 ng ml-1. The ELISA was used in bioassays for the detection of environmental estrogens, including estrogen mimics, in whole and fractionated industrial waste waters. Those bioassays were based on intraperitoneal (i.p.) injection-, static renewal-, and flow through exposure systems. The response threshold of both bioassays is limited to 1-2 micrograms ml-1 Vg by a low level plasma interference that was regularly detected in plasma from non-induced male fish. The responsiveness of the bioassays was characterized using progressive doses of 17 beta-estradiol. The i.p.-based assay, which was responsive to at least 100 micrograms kg-1 of 17 beta-estradiol, was used to screen extracts of pulp mill effluent and black liquor for estrogenic effects. Neither extract induced Vg in our assay. The i.p. assay was also used to test 4-tert-octylphenol (OP) and the PAH derivative, retene, for estrogenic activity. OP induced Vg in the i.p.-exposed fish; no Vg induction was detected in the retene-exposed fish. The static renewal bioassay, which was responsive to at least 0.1 microgram ml-1 of 17 beta-estradiol over a 15-day exposure period, was used to screen whole pulp mill effluents for estrogenic effects. No Vg induction was detected in the effluent-treated fish.     

FasL induces Fas/Apo1-mediated apoptosis in human embryonic kidney 293 cells routinely used to generate E1-deleted adenoviral vectors

Phosphatidylinositol-4-phosphate 5-kinase (PIP5K) phosphorylates phosphatidylinositol-4-phosphate to produce phosphatidylinositol-4,5-bisphosphate as a precursor of two second messengers, inositol-1,4,5-triphosphate and diacylglycerol, and as a regulator of many cellular proteins involved in signal transduction and cytoskeletal organization. Despite PIP5K playing such an essential role in a number of physiological processes, much still remains to be made clear about its association with plants. Searching the Arabidopsis expression sequence tag database against already known yeast and mammalian PIP5K cDNAs, we identified two clones which partly encode the same Arabidopsis PIP5K and isolated a corresponding full-length cDNA encoding a protein that we designated AtPIP5K1. Recombinant AtPIP5K1 expressed in Escherichia coli possessed a PIP5K activity in vitro. Due to some structural and biochemical differences, AtPIP5K1 was not categorized as either a type I or type II PIP5K. The expression of the AtPIP5K1 mRNA was induced rapidly by treating Arabidopsis plants with drought, salt and abscisic acid, which suggests that AtPIP5K11 is involved in water-stress signal transduction. These data give evidence for a close link between phosphoinositide signaling cascades and water-stress responses in plants.     

Renal gunshot wounds: methods of salvage and reconstruction

Over the past 14 years, 2079 patients have been seen at our institution with renal trauma. Of these, 84 sustained gunshot wounds (81 unilateral, 3 bilateral; a total of 87 renal units). We evaluated this group to characterize the nature of their injuries and establish a methodology for renal salvage and reconstruction. Preoperative radiographic staging was performed with excretory urography (IVP) or computed tomographic (CT) scanning. The injuries were classified into five categories: 16 contusions (18.4%), 12 minor lacerations (13.8%), 44 major lacerations (50.5%), six vascular injuries (6.9%), and nine combination laceration and vascular injury (10.3%). Most patients had multiple organ injuries, with 79 requiring associated surgical procedures (94%). The mean injury Severity Score (ISS) was 26.7 (range, 4-59). Based on radiographic and clinical staging criteria, 69 renal injuries were surgically explored (79.3%), and 12 patients underwent nephrectomy (13.8%). Forty-six renal units were reconstructed (66.6%) by various methods, including renorrhaphy, omental pedical flaps, mesh or peritoneal patch grafts, partial nephrectomy, and vascular repair. Overall, 75 renal units were salvaged (86.2%). Early renal vascular control was achieved in all patients who underwent renal exploration. Follow-up functional studies were done in 24 (28.5%): none had delayed nephrectomy or postinjury hypertension. Overall, 79 patients survived (94%); however, mortality was not related to renal injury. These findings suggest that aggressive radiographic staging coupled with early vascular control and careful selection of reconstructive techniques can ensure a high renal salvage rate in patients with renal gunshot injuries.