Association of Mucoid Encapsulated Moraxella duplex var. nonliquefaciens with Chronic Bronchitis

The recovery of two strains of highly mucoid encapsulated Moraxella duplex var. nonliquefaciens from the sputum of two patients suffering from chronic bronchitis is described. The biochemical, morphological, and pathogenic characteristics of this microorganism are discussed.     

Sarcolipin, the "proteolipid" of skeletal muscle sarcoplasmic reticulum, is a unique, amphipathic, 31-residue peptide.

The sarcoplasmic reticulum of rabbit skeletal muscle contains a small "proteolipid," i.e., a protein which is soluble in acidic CHCl3/CH3OH. We propose the name sarcolipin for this small protein, to signify its lipid-like solubility and association with the sarcoplasmic reticulum. We have determined the following amino acid sequence for sarcolipin, using protein chemistry methods: M E R S T R E L C L N F T V V L I T V I L I W L L V R S Y Q Y. This 31-residue sequence includes a 19-residue hydrophobic segment which probably spans the sarcoplasmic reticulum membrane. The molecular weight calculated from the sequence, 3733, agrees with that measured by fast atom bombardment mass spectrometry, showing that sarcolipin contains no attached fatty acyl or other prosthetic groups.     

Studies of individual carbon sites of hen egg white lysozyme by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Assignment of the nonprotonated aromatic carbon resonances to specific residues in the sequence.

The resonances of nonprotonated aromatic carbons in natural abundance 13C NMR spectra of hen egg white lysozyme are assigned to specific residues of the amino acid sequence. Chemical shift considerations, the effect of pH, and partially relaxed Fourier transform NMR spectra are used to assign each resonance to one of the seven types of nonprotonated aromatic carbons of amino acid residues. Spectra of chemically modified lysozyme samples yield various assignments to specific residues in the sequence. Line-broadening effects caused by binding of the relaxation probes Gd3+ and 4-N-acetamido-2,2,6,6-tetramethylipiperidine-1-oxyl yield specific assignments which are fully consistent with those based on chemical modifications. The effects of paramagnetic shift reagents and amino sugar inhibitors do not yield any obvious specific assignments. The effect of pH on the chemical shift of Cgamma of His-15 yields a pKalpha in agreement with published values, and indicates that the imidazole form of His-15 exists mainly (or entirely) as the Nepsilon3-H tautomer. The effect of pH on the chemical shifts (measured up to pH 8.8, at 38 degrees) of Czeta and Cgamma of the 3 tyrosine residues yields crude pKalpha values of 9.5 and 10 for Tyr-23 and one of the other tyrosines, respectively. The 3rd tyrosine residue does not exhibit titration behavior.     

Studies of chemically modified histidine residues of proteins by carbon 13 nuclear magnetic resonance spectroscopy. Reaction of hen egg white lysozyme with iodoacetate.

It is shown that natural abundance 13C NMR spectroscopy can be used to determine the structures and relative amounts of chemically modified forms of a histidine residue of a peptide or protein. The unfractionated product of the reaction of N alpha-acetyl-L-histidine with bromoacetate yields four resonances of nonprotonated aromatic carbons. These resonances are assigned (on a one-to-one basis) to C gamma of the intact amino acid, the two monocarboxymethylated derivatives (at N delta1 and N epsilon2), and the dicarboxymethylated derivative. The effect of pH on the chemical shift of C gamma is characteristic for each of the four species. This property is used to study the carboxymethylation of His-15 of hen egg white lysozyme upon treatment with iodoacetate. With the use of various reaction conditions, His 15 is carboxymethylated in detectable quantities only at N epsilon2. The spectra of the various reaction mixtures indicate which conditions are best for maximizing the yield of this derivative. A comparison of the spectrum of chromatographically pure [N epsilon2-carboxymethylhistidine-15]lysozyme with that of the intact protein indicates that the chemical modification does not significantly affect the conformation of the protein (at least in the regions of all aromatic amino acid residues).     

The effect of ionomycin on calcium fluxes in sarcoplasmic reticulum vesicles and liposomes.

Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.     

Titration behavior of individual tyrosine residues of myoglobins from sperm whale, horse, and red kangaroo.

The titration behavior of individual tyrosine residues of myoglobins has been studied by observing the pH dependence of the chemical shifts of Czeta and Cgamma of these residues in natural abundance of 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes, at 37 degrees) of cyanoferrimyoglobins from sperm whale, horse, and red kangaroo. A comparison of the pH dependence of the spectra of the three proteins yielded specific assignments for the resonance of Tyr-151 (sperm whale) and Tyr-103 (sperm whale and horse). Selective proton decoupling yielded specific assignments for Czeta of Tyr-146 of the cyanoferrimyoglobins from horse and kangaroo, but not the corresponding assignment for sperm whale. The pH dependence of the chemical shifts indicated that only Tyr-151 and Tyr-103 are titratable tyrosine residues. Even at pH 12, Tyr-146 did not begin to titrate. The titration behavior of C zeta and Cgamma of Tyr-151 of sperm whale cyanoferrimyoglobin yielded a single pK value of 10.6. The pH dependence of the chemical shift of each of the resonances of Tyr-103 of the cyanoferrimyoglobins from horse and sperm whale could not be fitted with the use of a single pK value, but was consistent with two pK values (about 9.8 and 11.6). Furthermore, the resonances of Czeta and Cgamma of Tyr-103 broadened at high pH. The titration behavior of the tyrosines of sperm whale carbon monoxide myoglobin and horse ferrimyoglobin was also examined. A comparison of all the experimental results indicated that Tyr-151 is exposed to solvent, Tyr-146 is not exposed, and Tyr-103 exhibits intermediate behavior. These results for myoglobins in solution are consistent with expectations based on the crystal structure.     

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments.

Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species...     

L'effetto Di Popolazione Nelle Colture Del Polline: Alcuni Aspetti Della Sua Natura

Abstract

Pollen population effect in artificial culture: some aspect of its nature. — The ability of pollen to grow on several rather different artificial media has not yet allowed investigators to determine the best conditions for each species. However a common requirement for all kinds of pollen is a high density of pollen grains on the culture medium, that stimulates the pollen germination and the pollen tube growth. Many investigators dealing with the causes of that phenomenon failed to reach a convincing conclusion on its causes. Our experiment was set to test the ability of an unknown pollen growth factor (PGF) to diffuse from growing pollen into the medium. Consequently after removal of the pollen culture a second pollen population grown on the same medium should be able to use the PGF eventually dissolved in it. Widely scattered populations of Malus pollen were grown onto membrane filters in contact with such «preconditioned» media and tested for their ability to germinate. Because the percentage of germination were lower than expected, wide-pored membranes (8.0 μ instead of 0.8 μ) were tested, thus achieving a rate of germination as high as that of the crowded populations of pollen. Hence it was argued that the PGF is water soluble, but is absorded on the membrane before reaching the contact of the pollen. However if the pores surface is small enough to be saturated by part of the PGF the free remainder is still enough to stimulate the small population of pollen. To test the assumption (BREWBAKER and KWACK, 1963) that simply Ca++ supported by other cations is the PGF, Ca++, Mg++ and K+ have been supplemented to the Standard medium as sulfates at a 10—⁴ M concentration, combined in a factorial design. The results are reported in table 2 and can be briefly summarized as follow. The most efficient ion appears to be K+, followed by Ca++ and Mg++. Each one is able to increase significantly the rate of germination with respect to the following ion, but two negative interactions (Ca × K and Mg × K, P: 0.01) were found to be significant. Because the only significant interactions were with K+, and because the mixtures containing K+ significantly differ but from treatment with Ca++ and Mg++, and not from K+, it could be tentatively stated that the negative interaction is at expenses of the K+ stimulatory effect (see fig. 1). The joint evaluation of both experiments strongly suggests the idea that the PGF is an organic rather than an inorganic molecule, with high affinity with the membrane on which it can be adsorbed. Most probably this organic growth factor needs an inorganic ion to display its stimulatory effect, but this cannot be identified with the PGF. In fact none of the tested inorganic molecules is adsorbed on the narrow pored membrane and they display full stimulatory activity through it. The second experiment seems to suggest the idea that such inorganic ion is K+.