Stability investigation of a cryo soft x-ray microscope by fiber interferometry.

We present a stability investigation of the Stockholm laboratory cryo soft x-ray microscope. The microscope operates at a wavelength of 2.48 nm and can image biological samples at liquid-nitrogen temperatures in order to mitigate radiation damage. We measured the stability of the two most critical components, sample holder and optics holder, in vacuo and at cryo temperatures at both short and long time scales with a fiber interferometer. Results revealed vibrations in the kHz range, originating mainly from a turbo pump, as well as long term drifts in connection with temperature fluctuations. With improvements in the microscope, earlier stability issues vanished and close-to diffraction-limited imaging could be achieved. Moreover, our investigation shows that fiber interferometers are a powerful tool in order to investigate position-sensitive setups at the nanometer level.

Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules.

Intercellular connexin channels (gap junction channels) have long been thought to mediate molecular signaling between cells, but the nature of the signaling has been unclear. This study shows that connexin channels from native tissue have selective permeabilities, partially based on pore diameter, that discriminate among cytoplasmic second messenger molecules. Permeability was assessed by measurement of selective loss/retention of tracers from liposomes containing reconstituted connexin channels. The tracers employed were tritiated cyclic nucleotides and a series of oligomaltosaccharides derivatized with a small uncharged fluorescent moiety. The data define different size cut-off limits for permeability through homomeric connexin-32 channels and through heteromeric connexin-32/connexin-26 channels. Connexin-26 contributes to a narrowed pore. Both cAMP and cGMP were permeable through the homomeric connexin-32 channels. cAMP was permeable through only a fraction of the heteromeric channels. Surprisingly, cGMP was permeable through a substantially greater fraction of the heteromeric channels than was cAMP. The data suggest that isoform stoichiometry and/or arrangement within a connexin channel determines whether cyclic nucleotides can permeate, and which ones. This is the first evidence for connexin-specific selectivity among biological signaling molecules.

Matrix-assisted ultraviolet laser desorption/ionization mass spectrometry applied to multiple forms of lipases.

Matrix-assisted ultraviolet laser desorption/ionization mass spectrometry was used to investigate heterogeneous patterns and molecular masses of microbial lipases from Penicillium camembertii, Geotrichum candidum, and Pseudomonas sp. Mass spectral peaks of the native, glycosylated lipases from P. camembertii and G. candidum were broader than those of the corresponding deglycosylated enzymes, indicative of heterogeneous glycosylations. The broader peaks in the mass spectra were caused by an overlapping of unresolved peaks, derived from single glycoprotein species. Molecular masses determined for the deglycosylated proteins were in excellent agreement with those deduced from amino acid composition and sequence data, whereas with conventional biochemical methods (gelfiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis) only very rough estimations of molecular masses were possible. By mass spectrometric analysis of the four fractions of chromatographically separated P. camembertii lipase molecular masses of 29,990, 34,030, 31,990, and 32,140 Da were found before and 29,960, 29,980, 29,990 and 30,010 Da, respectively, after deglycosylation. Thus from the four native fractions of P. camembertii lipase three were glycoproteins. G. candidum lipase showed an average molecular mass of 63,500 Da for the heterogeneously deglycosylated native form and a molecular mass of 59,650 Da for the deglycosylated enzyme. For the Pseudomonas lipase, which could only be isolated with lipids firmly attached, a molecular mass of 32,890 Da was determined, in close agreement with that derived from the cDNA sequence.

Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data.

A procedure for the purification of a very hydrophobic lipase from Pseudomonas sp. strain ATCC 21808 was elaborated by avoiding the use of long-chain detergents in view of subsequent crystallization of the enzyme. The purification procedure included chromatography on Q-Sepharose in the presence of n-octyl-beta-D-glucopyranoside, Ca2+ precipitation of fatty acids, and Octyl-Sepharose chromatography. The enzyme was purified 260-fold to a yield of 35% and a specific activity of 3,300 U/mg. The molecular weight was determined as 35,000; a polyacrylamide gel under nondenaturing conditions revealed a band at 110,000, and the isoelectric point proved to be at 4.5 to 4.6. The lipase crystallized with different salts and ethylene glycol polymers in the presence of n-octyl-beta-D-glucopyranoside and one alkyloligooxyethylene compound (CxEy) in the range from C5E2 to C8E4. The crystals diffract to a resolution of about 0.25 nm. Precession photographs revealed that they belong to space group C2 with lattice constants of a = 9.27 nm, b = 4.74 nm, c = 8.65 nm, and beta = 122.3 degrees, indicating a cell content of one molecule per asymmetric unit of the crystal. In hydrolysis of triglycerides, the lipase showed substrate specificity for saturated fatty acids from C6 to C12 and unsaturated long-chain fatty acids. Monoglycerides were hydrolyzed very slowly. The N-terminal sequence is identical to that of the lipase from Pseudomonas cepacia. Treatment with diethyl-p-nitrophenylphosphate affected the activities toward triolein and p-nitrophenylacetate to the same extent and with the same velocity.

[Human leukocyte antigen (HLA) complex and anti-thyroidal antibodies in Graves' disease]. / Uklad HLA a przeciwciala przeciwtarczycowe w chorobie Gravesa-Basedowa.

The study aimed at: 1) assessing occurrence of HLA-A, HLA-B and HLA-C antigens in patients with Graves' disease in comparison with control group of healthy individuals; 2) determining relationship between circulating serum antimicrosomal and antithyroglobulin antibodies and selected HLA complex antigens. Human leukocyte antigens A, B, and C were detected with serological technique using cytotoxicity test. Thyroidal antimicrosomal and antithyroglobulin antibodies were titrated with radioimmunological solid phase technique while anti-membrane antibodies with immunoenzyme technique. The study involved 50 patients with Graves' disease and 50 healthy individuals. HLA-B8, HLA-B15, HLA-B35, and HLA-Cw3 antibodies were detected more frequently in patients with Graves' disease than in the healthy individuals. Antimicrosomal and antithyroglobulin antibodies were detected in the same group in 76% and 58% of patients, respectively whereas anti-membrane antibodies in 92% of patients. Comparison of the occurrence of thyroidal antimicrosomal and antithyroglobulin antibodies with the presence of HLA-B8, HLA-B35, HLA-B15, and HLA-Cw3 antigens did not show statistically significant correlation between these two parameters.

Interaction of the pore forming-peptide antibiotics Pep 5, nisin and subtilin with non-energized liposomes.

The cationic peptide antibiotics Pep 5, nisin and subtilin depolarize bacterial and artificial membranes by formation of voltage-dependent multi-state pores. Studies with non-energized liposomes indicated that the peptides do not span the membrane in the absence of a membrane potential. The effects of Pep 5 and nisin on neutral membranes, as studied by membrane fluidity, phase transition points and carboxyfluorescein efflux, were small compared to melittin. Acidic liposomes were affected more strongly, indicative of primarily electrostatic interactions with phospholipid head groups. Subtilin may slightly enter the hydrophobic core as suggested by tryptophan fluorescence quenching and liposome fusion experiments.

Mode of action of the staphylococcinlike peptide Pep 5: voltage-dependent depolarization of bacterial and artificial membranes.

The cationic staphylococcinlike peptide Pep 5 is shown to depolarize bacterial and planar lipid membranes in a voltage-dependent manner. An artificial valinomycin-induced potassium diffusion potential across the cytoplasmic membrane of Staphylococcus cohnii 22 was sufficient to promote Pep 5 action. Thus, evidence is provided that a membrane potential of sufficient magnitude is the only prerequisite for Pep 5 activity. The voltage dependence was elucidated by macroscopic conductance measurements with black lipid membranes. A threshold potential of about -90 to -100 mV, which was deduced from experiments with bacterial cells, could be confirmed. Single pores were resolved which often occur as short-lived bursts and fluctuate among different conductance levels. Pore diameters were calculated ranging from 0.1 to 1 nm. Succinylation of the lysine residues of Pep 5 resulted in prolonged pore lifetimes and maintenance of distinct conductance levels. However, the succinylated peptide required a higher threshold potential, approximately -150 mV, than the native peptide, which is probably the reason for the reduced activity of the modified peptide against intact gram-positive bacteria.

Different mechanisms of insensitivity to the staphylococcin-like peptide Pep 5.

Three different mechanisms of insensitivity to the bactericidal membrane disrupting action of the staphylococcin-like peptide Pep 5 were found to occur among certain Gram-positive bacteria. i) The immunity of the producer of Pep 5 Staphylococcus epidermidis 5 was shown to be due to a Pep 5 antagonist, which was excreted along with Pep 5 during growth. The cells were killed by Pep 5 when incubated with high doses exceeding the concentration of the antagonist. ii) Bacillus subtilis W23 produced a protease which cleaved Pep 5 into fragments with greatly reduced bactericidal activity. Like with S. epidermidis 5, cells of B. subtilis were susceptible to the membrane damaging action of the peptide and growth in culture was only possible after inactivation of Pep 5. iii) By prolonged incubation in the presence of Pep 5 resistant mutants of S. cohnii 22 and S. epidermidis 5 Pep 5- could be selected. Their cytoplasmic membrane was not sufficiently disrupted by the peptide to promote active killing, resulting in unhindered growth of these mutants in the presence of high doses (200 AU/ml) of Pep 5.

Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin.

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca.-100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multi-state pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2-1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.