Interaction of the major epitope region of HIV protein gp41 with membrane model systems. A fluorescence spectroscopy study

Fluorescence spectroscopy (both steady-state and time-resolved) was used to study the fragment 579-601 of gp41 ectodomain (HIV-1), a highly conserved sequence and major epitope, regarding (1) structural information, (2) interaction with membrane model systems, and (3) location in the phospholipid bilayer. The peptide was characterized both in its monomeric (after reduction of the disulfide bond between cysteine residues) and in the dimeric forms. The change of the fluorescence anisotropy between monomer and dimer was rationalized on the basis of energy migration, and a distance between the two tryptophan (Trp) residues of approximately 6 A was obtained. Using different fluorescence spectroscopy approaches, it was demonstrated that, despite the fact that monomeric gp41 fragment incorporates in the membrane model systems studied, the dimeric form does not interact with these vesicles. A methodology based on the increase of the mean fluorescence lifetime averaged by the preexponentials was derived, to obtain the partition coefficient of the peptide in the different lipid systems. Fluorescence quenching using lipophilic probes and red edge excitation shift (REES) were used to study the location of the gp41 fragment in the membrane. It was concluded that the Trp residue is located in a shallow position, near the interface. The REES results show an uncommonly large wavelength shift (18 nm) for the gp41 fragment incorporated in the membrane. Our results are consistent with a "two steps" model for the gp41 fusion mechanism similar to the one proposed for influenza virus hemagglutinin.     

Electrical stimulation of human tibialis anterior: (A) contractile properties are stable over a range of submaximal voltages; (B) high- and low-frequency fatigue are inducible and reliably assessable at submaximal voltages

OBJECTIVES: To investigate the validity and reliability of submaximal voltage stimulation for assessing the 'fresh' contractile properties of human tibialis anterior muscle (TA) and the efficacy of such stimulation in inducing and assessing high- and low-frequency fatigue. INTERVENTIONS: (A) Contractile properties of fresh TA were assessed in six normal volunteers using multifrequency stimulation trains (comprising 2 seconds at each of 10, 20 and 50 Hz, arranged contiguously) over a range of submaximal voltages. (B) On three separate occasions, fatigue was induced in the TA of 10 normal volunteers by means of a 3-minute unbroken sequence of the described multifrequency stimulation trains, delivered at a 'standardized' submaximal voltage. This fatiguing protocol was preceded by discrete multifrequency stimulation trains, at the same standardized voltage, but followed by discrete multifrequency trains delivered over a range of submaximal voltages (which included the standardized voltage). OUTCOME MEASURES: In experiment A the 10:50 Hz and 20:50 Hz force ratios were analysed for between-voltages variability using coefficients of variation (CVs), and for trends using Friedman tests and post-hoc Wilcoxon tests. In experiment B low-frequency fatigue was detected using 10:50 Hz and 20:50 Hz force ratios derived from the discrete multifrequency trains. High-frequency fatigue was calculated from the decline in high-frequency force which occurred during the fatiguing protocol itself. Each parameter was assessed for between-days repeatability using CVs. RESULTS: In experiment A the 'fresh' 10:50 Hz force ratio was clearly unreliable at voltages which generated <10% of maximal voluntary contractile force (MVC) (CV< or =29.7%), but was reasonably reliable at voltages which generated 20-30% of MVC (CV < or = 11.5%; p = 0.847). The 'fresh' 20:50 Hz force ratio was,in contrast, extremely reliable throughout the tested voltage range (CV< or =5.8%; p = 0.636) in fresh muscle. In experiment B paired t-tests indicated that the fatiguing protocol induced significant high-frequency fatigue (p <0.0037) and low-frequency fatigue (p <0.0008 for 'fresh' versus 'fatigued' 10:50 Hz force ratio; p <0.0001 for 'fresh' versus 'fatigued' 20:50 Hz force ratio). In muscle thus fatigued, the 20:50 Hz force ratio was extremely reliable in the 20-33% of MVC range (CV < or =7.3%; p = 0.847). Between-days repeatability was poor for the 10:50 Hz force ratio in both fresh and fatigued muscle (CV < or =23.8 and 44.4% respectively), but was highly acceptable for both voluntary and stimulated fatigue indices and for the 20:50 Hz force ratio, the latter in both fresh and fatigued muscle. CONCLUSIONS: These results confirm the validity and reliability of submaximal voltages in assessing contractile properties (including low-frequency fatiguability) and inducing fatigue of human TA.     

Advantages of 2'-O-methyl oligoribonucleotide probes for detecting RNA targets

We have compared various kinetic and melting properties of oligoribonucleotide probes containing 2'-O-methylnucleotides or 2'-deoxynucleotides with regard to their use in assays for the detection of nucleic acid targets. 2'-O-Methyl oligoribonucleotide probes bound to RNA targets faster and with much higher melting temperatures (Tm values) than corresponding 2'-deoxy oligoribonucleotide probes at all lengths tested (8-26 bases). Tm values of both probes increased with length up to approximately 19 bases, with maximal differences in Tm between 2'-O-methyl and 2'-deoxy oligoribonucleotide probes observed at lengths of 16 bases or less. In contrast to RNA targets, 2'-O-methyl oligoribonucleotide probes bound more slowly and with the same Tm to DNA targets as corresponding 2'-deoxy oligoribonucleotide probes. Because of their greatly enhanced Tm when bound to RNA, 2'-O-methyl oligoribonucleotide probes can efficiently bind to double-stranded regions of structured RNA molecules. A 17 base 2'-O-methyl oligoribonucleotide probe was able to bind a double-stranded region of rRNA whereas the same 17 base 2'- deoxy oligoribonucleotide probe did not. Due to their enhanced Tm when bound to RNA targets, shorter 2'-O-methyl oligoribonucleotide probes can be used in assays in place of longer 2'-deoxy oligoribonucleotide probes, resulting in enhanced discrimination between matched and mismatched RNA targets. A 12 base 2'-O-methyl oligoribonucleotide probe had the same Tm as a 19 base 2'-deoxy oligoribonucleotide probe when bound to a matched RNA target but exhibited a much larger decrease in Tm than the 2'-deoxy oligoribonucleotide probe when bound to an RNA target containing either 1 or 2 mismatched bases. The increased Tm, faster kinetics of hybridization, ability to bind to structured targets and increased specificity of 2'-O-methyl oligoribonucleotide probes render them superior to corresponding 2'-deoxy oligoribonucleotides for use in assays that detect RNA targets.     

Novel hepatoselective insulin analogues: studies with covalently linked thyroxyl-insulin complexes

To explore the possibility that insulin analogues designed to have restricted access to peripheral tissues may display relative hepatoselectivity in vivo, Nalphabeta1-thyroxyl-insulin (B1-T4-Ins) and Nalphabeta1-thyroxyl-aminohexanoyl insulin (B1-T4-AHA-Ins) were synthesized. These insulin analogues bind thyroid hormone binding proteins to form high molecular weight complexes. Effects of intravenous infusions of B1-T4-Ins; B1-T4-AHA-Ins; combined thyroxine binding globulin (TBG) and B1-T4-Ins and combined TBG and B1-T4-AHA-Ins were compared with those of insulin infusion in hyperinsulinaemic euglycaemic clamp protocols in anaesthetized beagles (n=4 and n=3 for combined TBG infusions). Glucose turnover rates were measured using D-[3-3H]glucose infusion. With all 5 protocols the rate of glucose disappearance (Rd) was increased and the rate of endogenous glucose production (Ra) decreased from basal level 13.53+/-0.60 micromol kg(-1) min(-1)(p<0.05). Insulin-like activity for Ra and Rd was calculated as the area between the basal values of each variable and the subsequent values plotted graphically against time (AUC). For insulin, B1-T4-Ins, B1-T4-AHA-Ins, combined infusions of TBG+B1-T4-Ins, and TBG+B1-T4-AHA-Ins, respectively, AUC for Rd values were 6.30+/-0.69, 3.35+/-0.53, 4.40+/-0.64, 2.82+/-0.40 and 3.46+/-0.95 (mmol kg(-1)), all analogue infusions being different from insulin (p<0.05). AUC for Rd was further reduced by addition of TBG to B1-T4-AHA-Ins (p<0.05). In contrast the effect of all analogues on AUC for Ra was similar to that of insulin. These observations are compatible with the suggestion that insulin analogues which bind to thyroid hormone binding proteins retain access to hepatic insulin receptors which primarily control Ra. The reduced peripheral insulin-like effect (Rd) could be due to reduced transcapillary access to peripheral insulin receptor sites.     

Large sub-pleural air cysts: an extreme form of pulmonary interstitial emphysema

Pulmonary interstitial emphysema is a well-documented complication of positive-pressure ventilation. However, the occurrence of large sub-pleural air cysts is a less well-known, extreme manifestation of this entity. We present here an infant who developed this complication of pulmonary barotrauma during cardiopulmonary resuscitative efforts.     

Warm ischemic tolerance in collapsed pulmonary grafts is limited to 1 hour

OBJECTIVE: To determine the length of warm ischemic tolerance in pulmonary grafts from non-heart-beating donors. SUMMARY BACKGROUND DATA: If lungs could be retrieved for transplant after circulatory arrest, the shortage of donors might be significantly alleviated. Great concern, however, exists about the length of tolerable warm ischemia before cold preservation of pulmonary grafts retrieved from such non-heart-beating donors. METHODS: The authors compared the influence of an increasing postmortem interval on graft function in an isolated, room air-ventilated rabbit lung model during blood reperfusion up to 4 hours. Four groups of cadavers (four animals per group) were studied. In group 1, lungs were immediately reperfused. In the other groups, cadavers with lungs deflated were left at room temperature for 1 hour (group 2), 2 hours (group 3), or 4 hours (group 4). RESULTS: Pulmonary vascular resistance was enhanced in all ischemic groups compared with the control group. An increase was noted with longer postmortem intervals in peak airway pressure and in weight gain. A concomitant decline was observed in the venoarterial oxygen pressure gradient caused by progressive edema formation, as reflected by the wet-to-dry weight ratio at the end of reperfusion. CONCLUSIONS: Warm ischemia resulted in increased pulmonary vascular resistance. Graft function in lungs retrieved 1 hour after death was not significantly worse than in nonischemic lungs. Therefore, 60 minutes of warm ischemia with the lung collapsed may be tolerated before cold storage. Further studies are necessary to investigate whether lungs retrieved from non-heart-beating donors will become a realistic alternative for transplant.     

Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries

Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.     

Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases

The 2.15-A resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3' to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3' phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.     

Solubilization of hydrophobic peptides by reversible cysteine PEGylation

"PEG-a-Cys" reagent, synthesized by the esterification of monomethoxy-poly(ethylene glycol) (avg. MW = 5 kDa) to Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid)], is shown to "PEGylate" reversibly the cysteine residue of a 25-residue synthetic hydrophobic peptide (H2N-REAAALAAAAALAAWAALCPARRRR-CO2H) designed to model a transmembrane segment of a membrane protein. A mixed disulfide bond was formed between the reagent and the peptide that was readily cleaved with the mild reducing agent tricarboxyethylphosphine hydrochloride (TCEP.HCl). Carboxypeptidase B digestion of the charged carboxyl terminus of the peptide through to the Ala residue--which mimics the enzymatic cleavage of a TM segment from a fusion protein--releases a highly hydrophobic peptide. A time-dependent decrease in the amplitude of the digested peptide circular dichroism (CD) spectra was attributed to the aggregation and/or precipitation of the peptide. While PEGylation of the peptide with PEG-a-Cys had a negligible effect on conformation, it inhibited the loss of CD amplitude in both intact and digested peptides, suggesting that it was effective in solubilization of hydrophobic peptides.     

Telomerase activity and telomere lengths in various cell lines: changes of telomerase activity can be another method for chemosensitivity evaluation

For the cancer cells which have overcome the second mitotic clock (M2), activated telomerase is essential and used as another marker of immortality. Many trials had been initiated to target telomerase, which is known to be specific to tumors. To determine the best in vitro cell system for testing the efficacy of telomerase inhibitors, we evaluated the telomerase activity of various cancer cell lines and measured their telomere lengths. We also treated some cancer cell lines with adriamycin and measured the changes of telomerase activity. Telomerase activity was evaluated in various cell lines with the TRAP (telomeric repeat amplification protocol) assay. Telomerase activity was calculated and translated into arbitrary units by computer-assisted densitometry with the control of telomerase activity in the 293 control cell line. Also, terminal restriction fragment lengths were measured using Southern blotting. We also measured telomerase activity and telomere lengths in 11 benign breast tumor tissues and 19 paired stomach cancer and normal tissues. Cancer cell lines treated with adriamycin we evaluated for changes of telomerase activity and the cell proliferation by MTT assay and dye exclusion test. Telomerase activity of cell lines was 95.3 24.1 unit with a range of 27.6-129.6 unit, while the telomere lengths of those cell lines were variable from 5.0 to 10.4 kbp with a median of 6 kbp. In 11 cancer cell lines which were not yet firmly established, we could not detect any telomerase activity. Low telomerase activity was detected in only 2 benign tumor tissues of breast with a median telomere length of 8.8 (7-10.5) kbp. Among paired 19 gastric cancer and normal tissues, only 7 cancer tissues showed weak telomerase activity. After adriamycin treatment, telomerase activity in YCC-S-1, YCC-S-3, MCF-7 and MCF-7/ADR was decreased in accordance with the changes of the cell numbers. Telomerase is specific to cancer tissues and is expressed differently from organ to organ. Telomerase activity by TRAP assay could be used as a chemosensitivity assay.