Strategies for positioning fluorescent probes and crosslinkers on formyl peptide ligands

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1-3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.     

Hypoxia-ischemia induces a rapid elevation of ubiquitin conjugate levels and ubiquitin immunoreactivity in the immature rat brain

Postnatal rats at 7 and 21 days of age were subjected to unilateral hypoxia-ischemia (H/I) by right carotid artery ligation followed by 1.5 to 2 hours of hypoxia (8% oxygen). Brains were frozen at specific intervals of recovery from 0 to 24 hours. Western blots of samples of right and left forebrain were immunodeveloped with a monoclonal antibody specific for ubiquitin, RHUb1. An elevation of ubiquitin conjugate levels in the right compared with the left forebrain of 7-day-old animals was detectable immediately following H/I and increased by close to 60% of control level within 1 hour of recovery. The conjugate immunoreactivity remained at this level for 6 hours but had declined to control levels by 24 hours of recovery. No such increase was observed in response to hypoxia alone. Similar changes were observed in samples from the 21-day-old rat brain. However, the elevation of ubiquitin conjugate levels was of slower onset and persisted longer than observed for the 7-day-old animals. Immunocytochemical studies of brain fixed by immersion in formaldehyde/acetone/methanol showed that ubiquitin-like immunoreactivity was increased in the right, but not left, cerebral cortex and hippocampus of animals subjected to H/I. The data suggest that elevated ubiquitination may represent a neuroprotective response to H/I.     

Relationship between prothrombin activation fragment F1.2 and international normalized ratio in patients with atrial fibrillation. Stroke Prevention in Atrial Fibrillation Investigators

BACKGROUND AND PURPOSE: The prothrombin time (expressed as the international normalized ratio [INR]) is the standard method of monitoring warfarin therapy in patients with atrial fibrillation. Prothrombin activation fragment F1.2 provides an index of in vivo thrombin generation and might provide a better index of the effective intensity of anticoagulation. We examined the relationship between F1.2 and INR in patients with atrial fibrillation. METHODS: We measured INR and F1.2 levels in 846 patients with atrial fibrillation participating in the Stroke Prevention in Atrial Fibrillation III study. Two hundred nineteen (26%) were taking aspirin alone, 326 (39%) were taking adjusted-dose warfarin, and 301 (36%) were taking a low fixed dose of warfarin (1 to 3 mg) plus aspirin (combination therapy). F1.2 levels were measured with an enzyme-linked immunosorbent assay. RESULTS: Patients receiving adjusted-dose warfarin or combination therapy had significantly higher INR and significantly lower F1.2 values than those on aspirin alone (P < or = .0001 for each of the four comparisons). F1.2 values (nanomolar) were inversely correlated with INR (F1.2 = -0.1 + 2.3[1/INR]; R2 = .37; P < .0001; simple linear regression). However, significant variability remained. Among patients receiving warfarin, older patients had higher F1.2 values than younger patients after adjustment for INR intensity (P < .001) in the model. There was no difference in the relationship between F1.2 and INR between men and women. CONCLUSIONS: Increasing intensity of anticoagulation, as measured by the INR, is associated with decreasing thrombin generation as measured by the F1.2 level, but significant variability exists in this relationship. Older anticoagulated patients have higher F1.2 values than younger patients at equivalent INR values. The clinical significance of these differences is not clear. F1.2 measurement might provide information regarding anticoagulation intensity in addition to that reflected by the INR.     

Incisional hernia after laparoscopic nephrectomy with intact specimen removal: caveat emptor

The choline-containing phosphoglycolipid, MfGL-II, is the major polar lipid of Mycoplasma fermentans PG18. Anti-MfGL-II antisera raised in rabbits using the purified MfGL-II as an immunogen were employed in immunogold electron microscopic and immunofluorescence studies showing that MfGL-II is uniformly distributed and exposed on the cell surface of M. fermentans cells. The specificity of the antibodies was determined by immunostaining of lipid extracts separated by thin layer chromatography. The antibodies recognize lipids specific to M. fermentans but did not cross-react with lipid extracts of M. penetrans, M. capricolum, M. gallisepticum or Acholeplasma laidlawii. As phosphocholine almost completely abolished antibody interaction with MfGL-II in an ELISA assay it is suggested that the anti-MfGL-II repertoire is composed primarily of anti-phosphocholine antibodies. The anti-MfGL-II antisera inhibit the attachment of M. fermentans to Molt-3 lymphocytes suggesting that MfGL-II plays a major role in M. fermentans-host cell interaction.     

Analysis of transient permeation fluxes into and out of membranes for adsorption measurements

A relationship between the integrated fluxes into and out of a membrane following a positive or negative step change in feed concentration was derived. This analysis allows adsorption isotherms in the transport pathways through membranes to be determined from transient permeation responses to step concentration changes in the feed without measuring the retentate response. For Fickian diffusion through a membrane with Langmuir adsorption and zero coverage at the permeate boundary, the difference between the time-integrated flux into and flux out of the membrane is shown to be three times the time-integrated difference between the steady-state flux and the flux out. For Maxwell-Stefan diffusion, this ratio of integrated flux differences is 3 at low coverages and decreases towards 2 at saturation coverage. Mass transfer resistance at the permeate boundary increases the Fickian ratio above 3, and the ratio increases with decreasing Sherwood number. The ratio of integrated flux differences is shown to be identical to the steady-state replenishment time divided by the time lag. Thus, the ratio can be calculated directly from the steady-state concentration profile and the concentration-dependent diffusion coefficient. Surface diffusion through zeolite membranes was analyzed to demonstrate the calculation of the flux relationship for specific adsorption and diffusion models, but the method developed can be applied to membrane permeation in general.     

Incorporating a Polyethyleneglycol Linker to Enhance the Hydrophilicity of Mitochondria-Targeted Triphenylphosphonium Constructs

The targeting of bioactive molecules and probes to mitochondria can be achieved by coupling to the lipophilic triphenyl phosphonium (TPP) cation, which accumulates several hundred-fold within mitochondria in response to the mitochondrial membrane potential (Δψ_m). Typically, a simple alkane links the TPP to its “cargo”, increasing overall hydrophobicity. As it would be beneficial to enhance the water solubility of mitochondria-targeted compounds we explored the effects of replacing the alkyl linker with a polyethylene glycol (PEG). We found that the use of PEG led to compounds that were readily taken up by isolated mitochondria and by mitochondria inside cells. Within mitochondria the PEG linker greatly decreased adsorption of the TPP constructs to the matrix-facing face of the mitochondrial inner membrane. These findings will allow the distribution of mitochondria-targeted TPP compounds within mitochondria to be fine-tuned.     

Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant

Constrained binding peptides (peptide aptamers) may serve as tools to explore protein conformations and disrupt protein-protein interactions. The quality of the protein scaffold, by which the binding peptide is constrained and presented, is of crucial importance. SQT (Stefin A Quadruple Mutant-Tracy) is our most recent development in the Stefin A-derived scaffold series. Stefin A naturally uses three surfaces to interact with its targets. SQT tolerates peptide insertions at all three positions. Peptide aptamers in the SQT scaffold can be expressed in bacterial, yeast and human cells, and displayed as a fusion to truncated pIII on phage. Peptides that bind to CDK2 can show improved binding in protein microarrays when presented by the SQT scaffold. Yeast two-hybrid libraries have been screened for binders to the POZ domain of BCL-6 and to a peptide derived from PBP2', specific to methicillin-resistant Staphylococcus aureus. Presentation of the Noxa BH3 helix by SQT allows specific interaction with Mcl-1 in human cells. Together, our results show that Stefin A-derived scaffolds, including SQT, can be used for a variety of applications in cellular and molecular biology. We will henceforth refer to Stefin A-derived engineered proteins as Scannins.     

Transient measurements of adsorption and diffusion in H-ZSM-5 membranes

A transient permeation method presented here not only determines the adsorption and diffusion properties of the pores that are the transport pathways through zeolite membranes, but nondestructively estimates the effective thickness of the membrane. Transient responses of the permeate concentration to step changes in the feed were measured on two H-ZSM-5 tubular membranes and modeled assuming Maxwell-Stefan diffusion and Langmuir adsorption. The adsorption isotherms determined from these transient measurements at 298 K of N₂ and CO₂ were nearly identical to those measured by calorimetry on H-ZSM-5 powders. The CH₄ isotherm at 298 K was similar to isotherms measured by calorimetry and gravimetric techniques on Na-ZSM-5 and silicalite powders. The similarity of the isotherms indicates that transport of these light gases occurs mainly through zeolite pores. The Maxwell-Stefan diffusion coefficients D_MS depended on concentration and were higher for higher feed partial pressures. Average D_MS values for the two membranes were 7.5, 5 and 1.5×10⁻¹⁰ m²/s for N₂, CH₄, and CO₂, respectively; these are in the same range and order as diffusion coefficients measured in zeolite crystals.     

Development of a FACS-verified set of basic and self-conscious emotion expressions.

In 2 studies, the authors developed and validated of a new set of standardized emotion expressions, which they referred to as the University of California, Davis, Set of Emotion Expressions (UCDSEE). The precise components of each expression were verified using the Facial Action Coding System (FACS). The UCDSEE is the first FACS-verified set to include the three “self-conscious” emotions known to have recognizable expressions (embarrassment, pride, and shame), as well as the 6 previously established “basic” emotions (anger, disgust, fear, happiness, sadness, and surprise), all posed by the same 4 expressers (African and White males and females). This new set has numerous potential applications in future research on emotion and related topics. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The practice of psychological science: Searching for Cronbach’s two streams in social–personality psychology.

The present research surveyed a group of editors and editorial board members of personality and social psychology journals to examine the practice of psychological science in their field. Findings demonstrate that (a) although personality and social researchers tend to use many of the same approaches, methods, and procedures, they nonetheless show average differences in each of these domains, as well as in their overarching theoretical aims and perspectives; (b) these average differences largely conform to social and personality researchers’ stereotypes about each subgroup; (c) despite their methodological and philosophical differences, the 2 subgroups study many of the same research topics; and (d) the structure of social–personality research practices can be characterized as having 2 independent factors, which closely correspond to L. J. Cronbach’s (1957) correlational and experimental “streams of research.” (PsycINFO Database Record (c) 2010 APA, all rights reserved)