Effect of short-chain primary alcohols on fluidity and activity of sarcoplasmic reticulum membranes

Intramolecular excimer formation with the fluorescent probe 1,3-di(1-pyrenyl)propane, differential scanning calorimetry, and X-ray diffraction were used to assess the effect of ethanol, 1-butanol, and 1-hexanol on the bilayer organization in model membranes, sarcoplasmic reticulum (SR) lipids and native SR membranes. These alcohols have fluidizing effects on membranes and lower the main transition temperature of dimyristoylphosphatidylcholine (DMPC), but only 1-hexanol alters the cooperativity of the phase transition and significantly increases the thickness of DMPC bilayers. The interaction of the three alcohols with the SR Ca2+ pump was also investigated. Hydrolysis of ATP and coupled Ca2+ uptake are differently sensitive to the three alcohols. Whereas ethanol and 1-butanol inhibited the Ca2+ uptake, 1-hexanol stimulated it. Nevertheless, the energetic efficiency of the pump (Ca2+/ATP) is not significantly affected by ethanol or 1-hexanol, but uncoupling was observed with 1-butanol at high concentrations. The different effects of alcohols on the activity of SR membranes rule out an unitary mechanism of action on the basis of fluidity changes induced in the lipid bilayer. Depending on the chain length, the alcohols interact with the SR membranes in different domains, perturbing differently the Ca2+-pump activity.     

NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in reproductive parameters during the spawning migration of pink salmon, Oncorhynchus gorbuscha (Walbaum)

The hormones 17β-estradiol, 17α-hydroxy-20β-dihydroprogesterone(17α, 20β-P), 11-ketotestosterone, testosterone, gonadotropin and also vitellogenin, were determined during the spawning migration of wild pink salmon in the Fraser and Thompson Rivers in British Columbia. This stock of pink salmon takes approximately 2 weeks to migrate the 333 km upstream to the spawning grounds. Both sexes were at an advanced stage of sexual development when they entered fresh water. In females both the 17β-estradiol and vitellogenin levels fell precipitously during the migration, to be very low at spawning, whereas the 17α,20β-P level rose rapidly, to be highest at arrival on the spawning grounds. The gonadotropin level also rose rapidly during the migration, and was highest in spent fish. Testosterone was at a high level throughout, although this level decreased steadily during migration. In many respects similar endocrine changes were observed in the male. For example, in the case of androgen levels, both testosterone and 11-ketotestosterone fell steadily during migration but were still relatively high at spawning, whereas both gonadotropin and 17α, 20β-P levels rose markedly as migration progress. However, although the qualitative changes were often similar between the sexes, the levels of 17α, 20β-P, testosterone, and gonadotropin were considerably higher throughout in females than in males. It is concluded that this stock of pink salmon is at an advanced stage of sexual development when it enters fresh water. The endocrine changes observed during this study represent those controlling the final stages of reproduction, specifically final oocyte maturation and ovulation in females, and the final stages of spermatogenesis and spermiation in males.     

Recombinant human interleukin 1 alpha: purification and biological characterization

Interleukin 1 (IL 1) is a polypeptide hormone produced by activated macrophages that affects many different cell types involved in immune and inflammatory responses. The cloning and expression of a murine IL 1 cDNA in Escherichia coli encoding a polypeptide precursor of 270 amino acids has been reported, and expression of the carboxy-terminal 156 amino acids of this precursor in E. coli yields biologically active IL 1. By using the murine IL 1 cDNA as a probe, we have isolated its human homolog from cDNA generated to lipopolysaccharide-stimulated human leukocyte mRNA. Nucleotide sequence analysis of this cDNA predicts a protein of analysis of this cDNA predicts a protein of 271 amino acids (termed IL 1 alpha) which shows congruent to 61% homology to its murine counterpart but only 27% homology to a recently characterized human IL 1 precursor (IL 1 beta). We have expressed the carboxy-terminal 154 amino acids of IL 1 alpha in E. coli, purified this protein to homogeneity, and have compared it with pure recombinant murine IL 1 in several different IL 1 assays based on murine and human cells. Recombinant IL 1 is capable of stimulating T cell and fibroblast proliferation and inducing fibroblast collagenase and prostaglandin production, thus proving that a single molecule has many of the activities previously ascribed to only partially purified IL 1 preparations. Our results indicate that there exists a family of at least two human IL 1 genes (alpha and beta) whose dissimilar protein products have similar biological activities.     

Sodium channels in planar lipid bilayers. Channel gating kinetics of purified sodium channels modified by batrachotoxin

Single channel currents of sodium channels purified from rat brain and reconstituted into planar lipid bilayers were recorded. The kinetics of channel gating were investigated in the presence of batrachotoxin to eliminate inactivation and an analysis was conducted on membranes with a single active channel at any given time. Channel opening is favored by depolarization and is strongly voltage dependent. Probability density analysis of dwell times in the closed and open states of the channel indicates the occurrence of one open state and several distinct closed states in the voltage (V) range-120 mV less than or equal to V less than or equal to +120 mV. For V less than or equal to 0, the transition rates between stages are exponentially dependent on the applied voltage, as described in mouse neuroblastoma cells (Huang, L. M., N. Moran, and G. Ehrenstein. 1984. Biophysical Journal. 45:313-322). In contrast, for V greater than or equal to 0, the transition rates are virtually voltage independent. Autocorrelation analysis (Labarca, P., J. Rice, D. Fredkin, and M. Montal. 1985. Biophysical Journal. 47:469-478) shows that there is no correlation in the durations of successive open or closing events. Several kinetic schemes that are consistent with the experimental data are considered. This approach may provide information about the mechanism underlying the voltage dependence of channel activation.     

The ligand of the erythrocyte receptor of T lymphocytes: expression on white blood cells and possible involvement in T cell activation

We have recently described a monoclonal antibody (mAb) to sheep erythrocytes, termed L180/1, that blocks the formation of E rosettes between human or sheep T lymphocytes and sheep red blood cells. The cell surface glycoprotein (GP) of 42,000 apparent m.w. recognized by mAb L180/1 was given the preliminary name T11 target structure or T11TS. In the present report, it is shown that T11TS is also expressed on sheep white blood cells, notably on activated T lymphocytes, that are shown to actively synthesize this cell surface GP. In addition, the mixed lymphocyte reaction between outbred sheep is inhibited by mAb L180/1 at an early stage of the response. Together with the known involvement of the E receptor in T lymphocyte activation, these results are taken to suggest that T11 and T11TS are complementary cell interaction molecules involved in regulating T cell proliferation.     

Beta-lactamase producing Haemophili occurrence in clinical materials and carriage in healthy children

Beta-lactamase production was investigated in 126 H. influenzae and 15 H. parainfluenzae strains isolated in various infections. In H. influenzae the rate of beta-lactamase positive strains was 5.6%, in non-encapsulated strains it was higher (9.7%) than in capsule bearing strains (3.1%). Among beta-lactamase producing H. influenzae strains biotype II was predominant, whereas biotype I prevailed in beta-lactamase positive strains of H. parainfluenzae. A study undertaken in 101 children of a day-care nursery revealed 16.8% carriers of beta-lactamase producing Haemophili. Among the isolated strains we found the double number of H. parainfluenzae than H. influenzae strains showing beta-lactamase activity. This result supports the hypothesis of H. parainfluenzae being the reservoir of resistances plasmids in Haemophili.     

High performance liquid chromatography and time-of-flight secondary ion mass spectrometry: a new dimension in structural analysis of apolipoproteins

We report the isolation and characterization of an apolipoprotein A-I mutant using a new technique for structural analysis of apolipoproteins based upon the combined techniques of protein isolation by isoelectric focusing in immobilized pH-gradients, reversed-phase HPLC of tryptic peptides, and subsequent molecular weight analysis of isolated peptides by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The particular advantages of the TOF-SIMS procedure in the characterization of proteolytic peptides are the detection limits in the picomole range, the accuracy of molecular weight determination (up to 3000 +/- 1 D), the speed of analysis, and the wide range of applications for involatile biomolecules. The described procedure for the analysis of apolipoproteins requires only 2 ml of serum as starting material. This method can be used to monitor for genetic polymorphisms and posttranslational modifications on a microscale basis. Applying these techniques, we characterized a new apolipoprotein A-I mutant with an amino acid exchange arginine177 by histidine.