Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles

Infrared spectra were obtained as a function of temperature for a variety of phospholipid/water bilayer assemblies (80% water by weight) in the 3000-950 cm^?1 region. Spectral band-maximum frequency parameters were defined for the 2900 cm^?1 hydrocarbon chain methylene symmetric and asymmetric stretching vibrations. Temperature shifts for these band-maximum frequencies provided convenient probes for monitoring the phase transition behavior of both multilamellar liposomes and small diameter single-shell vesiclesof dipalmitoyl phosphatidylcholine/water dispersions. As examples of the effects of bilayer lipid/cholesterol/water (3 : 1 mol ratio) and lipid/cholesterol/amphotericin B/water (3 : 1 : 0.1 mol ratios) vesicles were examined using the methylene stretching frequency indices. In comparison to the pure vesicle form, the transition width of the lipid/cholesterol system increased by nearly a factor of two (to 8°C) while the phase transition temperature remained approximately the same (41° C). For the lipid/cholesterol/amphotericin B system, the phase transition temperature increased by about 4.5° C (to 45.5°C) with the transition width increasing by nearly a factor of four ($\text{to}\text{≈ 15°}\text{C}$) above that of the pure vesicles. The lipid/cholesterol/amphotericin B data were interpreted as reflecting the formation below 38°C of a cholesterol/amphotericin B complex whose dissociation at higher temperature (38–60°C range) significantly broades the gel-liquid crystalline phase transition.     

Thrombin- and histamine-induced signal transduction in human endothelial cells. Stimulation and agonist-dependent desensitization of protein phosphorylation

Treatment of human endothelial cells with thrombin, histamine, or dioctanoylglycerol (DiC8), a synthetic diacylglycerol, resulted in the rapid and transient phosphorylation of a Mr = 29,000 protein (P29) in a dose-dependent manner. Various tumor promoters also promoted P29 phosphorylation while the adenylate cyclase activator, forskolin, did not. The level of phosphorylation with all three agonists was similar (2.5-4 fold), and analysis of P29 by two-dimensional gel electrophoresis revealed identical patterns in each case. Receptor specificity was demonstrated for the histamine-stimulated changes; pyrilamine (10(-6) M; H1) but not cimetidine (10(-4); H2) blocked the response. The thrombin effect was active site-dependent. Phosphorylation induced by thrombin and histamine occurred within 1 min, peaked between 5 and 10 min, and returned to control levels by 1 h. DiC8-induced phosphorylation occurred more slowly but was also reduced by 1 h while phorbol ester treatment prolonged phosphorylation for at least 4 h. Treatment of these cells with thrombin or histamine for 1 h desensitized P29 to further phosphorylation by the homologous agonist although secondary phosphorylation could occur with heterologous compounds. However, if the primary agonist was removed following the onset of a desensitized state, secondary phosphorylation of P29 could be stimulated by the same compound. These same results were observed with two other phosphoproteins Mr = 18,000 (P18) and 80,000 (P80) which became more highly phosphorylated in response to thrombin treatment and with histamine/thrombin-stimulated prostaglandin I2 production. In contrast, homologous down-regulation of P29 phosphorylation was not observed with DiC8-treated cells, and the decline in phosphorylated P29 was associated with the loss of functional DiC8. The protein kinase inhibitors staurosporine and H-7 blocked P18 and P80 phosphorylation by thrombin but had no effect on P29 phosphorylation by histamine, thrombin, or DiC8 suggesting distinct pathways leading to the phosphorylation of these different proteins. These data suggest that multiple and independent thrombin/histamine-induced events are susceptible to receptor occupancy-dependent homologous down-regulation.     

The protein structure code: what is its present status?

Current methods of prediction of protein conformation are reviewedand the algorithms on which they rely are presented. For non-homologousproteins and after cross-validation the reported methods exhibita probability index, i.e. the per cent of correctly predictedresidues per predicted residues, of 63–65% with a standarddeviation of the order of 7% for three conformational states—helix,ß-strand and coil. This present limitation in theaccuracy of predictions that use only the information of thelocal sequence can be related essentially to the effect of long-rangeinteractions specific for each protein family. The methods basedon sequence similarity can improve the accuracy of predictionby expressing explicitly the homology of the protein to be predictedwith proteins in the database. In these circumstances the probabilityindex can reach 87% with a standard deviation of 6.6%. Thisproperty can be used for modeling homologous proteins by aidingin amino acid sequence alignments. The prediction of the tertiarystructure of a protein is still limited to the case of modelinga structure based on the known three-dimensional structure ofa homologous protein.     

Glycosylation and membrane insertion of newly synthesized rat dopamine beta-hydroxylase in a cell-free system without signal cleavage.

Dopamine beta-hydroxylase (DBH, EC 1.14.17.1) is present in both membrane-bound and soluble forms in neurosecretory vesicles. This study was designed to investigate the differences between membrane-bound and soluble DBH and how they may arise from translation of a single mRNA. Antisera to a peptide corresponding to the carboxyl terminus of rat DBH was found to specifically immunoprecipitate the 77- and 73-kDa subunits of newly synthesized DBH in rat brain. Thus, both soluble and membrane-bound forms contain the same carboxyl terminus. To investigate differences at the amino terminus, full-length rat DBH mRNA, translated in a cell-free system, produced a 66-kDa peptide. An additional higher molecular mass product was synthesized upon co-translational addition of microsomal membranes. This product was glycosylated since it bound to concanavalin A-Sepharose and reverted to the 66-kDa polypeptide after treatment with endoglycosidase H. This glycosylated product was resistant to protease digestion and fractionated with microsomal membranes on sucrose gradients, indicating that it is incorporated into the microsomal membranes. Amino-terminal sequencing of the glycosylated translation product indicated that the amino-terminal "signal" sequence was not cleaved. The results indicate that in the cell-free system newly synthesized DBH undergoes glycosylation and incorporation into microsomal membranes without cleavage of the NH2-terminal signal sequence.     

Raman spectroscopic studies of dimyristoylphosphatidic acid and its interactions with ferricytochrome c in cationic binary and ternary lipid-protein complexes.

The vibrational Raman spectra of both pure 1-alpha-dimyristoylphosphatidic acid (DMPA) liposomes and DMPA multilayers reconstituted with ferricytochrome c at pH 7 and pH 4, with either sodium or calcium as the cation, are reported as a function of temperature. Multilayers composed of a 1:1 mol ratio DMPA and dimyristoylphosphatidylcholine with perdeuterated acyl chains (DMPC-d54) have also been reconstituted with approximately 10(-4) M ferricytochrome c for Raman spectroscopic observation. Total integrated band intensities and relative peak height intensity ratios, two spectral Raman scattering parameters used to characterize bilayer properties, are sensitive to the presence of both ferricytochrome c and the cation in the reconstituted liposomes. Temperature profiles, derived from the various Raman intensity parameters for the 3,100-2,800 cm-1 lipid acyl chain C-H stretching mode region specifically reflect bilayer perturbations due to the interactions of ferricytochrome c. At pH 4 the calcium DMPA multilamellar gel to liquid crystalline phase transition temperatures Tm, defined by either the C-H stretching mode I2850/I2880 and I2935/I2880 peak height intensity ratios, are 58.5 +/- 0.5 degrees C and 60.0 +/- 0.3 degrees C, respectively. This difference in Tm's resolves the phase transition process into first an expansion of the lipid lattice and then a melting of the lipid acyl chains. At pH 7 the calcium DMPA liposomes show no distinct phase transition characteristics below 75 degrees C. For sodium DMPA liposomes reconstituted with ferricytochrome c at either pH 4.0 or pH 7.0, spontaneous Raman spectra show altered lipid structures at temperatures above 40 degrees C. Resonance Raman spectra indicate that ferricytochrome c reconstituted in either calcium or sodium DMPA liposomes changes irreversibly above Tm. For either the binary lipid or ternary lipid-protein systems reconstituted with DMPC-d54, linewidth parameters of the DMPC-d54 acyl chain CD2 symmetric stretching modes at 2,103 cm-1 provide a sensitive measure of the conformational and dynamic properties of the perdeuterated lipid component, while the 3,000 cm-1 C-H spectral region reflects the bilayer characteristics of the DMPA species in the complex. Although calcium clearly induces a lateral phase separation in the DMPA/DMPC-d54 system at pH 7.5 (Kouaouci, R., J.R. Silvius, I. Grah, and M. Pezolet. 1985. Biochemistry. 24:7132-7140), no distinct lateral segregation of the lipid components is observed in the mixed DMPA/DMPC-d54 lipid system in the presence of either ferricytochrome c or the sodium and calcium cations at pH 4.0.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recombination in the B Chromosome of Maize to Produce a-B-a Chromosomes

The translocations between the supernumerary B chromosomes and the normal A chromosomes of maize provide a valuable tool for gene localizations, dosage studies and characterization of mutants as null, leaky or gain-of-function. A procedure is described, that relies on recombination in the B chromosome, for marking each of the various B-A translocations with a single dominant marker that will allow dosage classifications of individuals at the mature kernel stage. This marker is R-scm3, which conditions anthocyanin pigment in the aleurone of the endosperm and the scutellum of the embryo. A test for recombination in the B chromosome was conducted by crossing together two translocations, that were broken on opposite sides of the B centromere, and in different A chromosome arms, namely TB-1La and TB-10L18. An example was recovered that linked genetic markers on 1L and 10L to the B centromere. Cytological examination at pachytene of meiosis confirmed the new chromosomal linkage. The use of this procedure to produce a comprehensive set of uniformly marked B-A translocations is discussed.     

Quantitative procedure for enumeration of bifidobacteria.

A membrane filter technique has been developed for the enumeration of bifidobacteria in natural aquatic environments. The technique is quantitative, selective, and differential. The medium (YN-6) contains: yeast extract, 2.0 g; agar, 1.5 g; polypeptone peptone, 1.0 g; vitamin-free Casamino Acids, 0.8 g; sodium chloride, 0.32 g; and L-cysteine hydrochloride, 0.003 g; in 100 ml of deionized water. The medium is adjusted to pH 7.0 before autoclaving. Nalidixic acid (80 micrograms/ml), neomycin sulfate (2.5 micrograms/ml), and bromcresol green (300 micrograms/ml) are included as selective and differential agents. After incubation for 48 h at 37 degrees C in an anaerobic environment, Gram-stained smears from green, glistening, smooth entire colonies are examined microscopically for typical bifidobacterial morphology. No significant difference in recoveries was observed when YN-6 was compared with reinforced clostridial agar, using bifidobacteria freshly isolated from feces and raw sewage. Using this technique with aquatic and fecal samples, less than 9% false-positive and 8% false-negative isolates were observed. These results indicated that the medium was able to satisfactorily recover organisms from a variety of situations.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.