Effects of alkali ions on myosin conformation

We have used two probes to study the effects of alkali ions on the conformation of myosin. One was paramagnetic, the “spin label” N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide, which binds primarily to SH groups; and the other was fluorescent, l-anilino-8-naphthalenesulfonate, which binds to an apolar niche. The bonding of the spin label to myosin was carried out in 0.6M LiCl, 0.6M NaCl, or 0.6M KCl, and the resulting labeled myosin was studied in the same medium in which the myosin was labeled as well as in other alkali chlorides. The electron paramagnetic resonance spectra of the spin label showed that the structure of myosin in the vicinity of the labeled groups differed in the various salts. The protein surface in the region of the labeled groups restricted the rotational freedom of the spin label more in KCl than in any of the other salts. Although ions are known to influence the properties of myosin, our results show that these ions also effect the molecular structure. The fluorescence of l-anilino-8-naphthalenesulfonate, noncovalently attached to myosin in the presence of alkali chlorides, decreased progressively with increasing size of the cations, again showing the protein structure near the probe attachment to be a function of the cation, in the solvent. Ca²⁺ quenched the fluorescence of the bound probe, indicating an interaction between Ca²⁺ and the myosin molecule. The effect of Ca²⁺ on the fluorescence was greatest in KCl.     

Chronic Pseudomonas aeruginosa endobronchitis in rhesus monkeys: II. A histopathologic analysis

We have recently established a rhesus monkey model of chronic Pseudomonas aeruginosa (PA) endobronchitis by bronchoscopic instillation of PA-embedded agar beads. All experimental animals developed chronic neutrophilic endobronchitis similar to chronic PA endobronchitis in cystic fibrosis (CF). Histopathologic studies further confirmed similarities to chronic PA endobronchitis in CF, including marked peribronchial inflammation, epithelial damage, presence of degraded cilia and ciliary abnormalities, appearance of PA bacterial clusters, mucosal hyperplasia, goblet cell hypertrophy/hypersecretion, airway obstruction, alveolar abnormalities, bronchiectasis, and fibrosis.     

Insertional inactivation of a chromosomal locus that modulates expression of potential virulence determinants in Staphylococcus aureus.

A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this locus is distinct from agr, xpr, and sar, three previously described global regulatory loci. Transduction experiments demonstrated that the genotype associated with mutant 1E3 could be transferred back into the parental strain ISP479C. The transductant 1E3-2 displayed a phenotypic profile similar to that of the original mutant. Northern (RNA) blot studies showed that this locus may be involved in modulating target genes at the mRNA level. In the rabbit endocarditis model, there was a significant decrease in both the infectivity rate and intravegetation bacterial density with mutant 1E3 compared with the parent at an inoculum of 10(3) CFU. Since protein A and the fibrinogen-binding protein(s) are major surface proteins that may mediate bacterial adhesion to host tissues, this locus may be an important genetic element involved in the expression of virulence determinants in S. aureus.     

Metabolism of progesterone in the pregnant sheep near term: identification of 3 beta-hydroxy-5 alpha-pregnan-20-one 3-sulfate as a major metabolite.

Two pregnant ewes near term were given a single injection of progesterone-4-14C via the left jugular vein, and serial blood samples were taken from the right jugular vein at 5 min intervals over a period of 40 min. Radioactive steroids in the plasma were separated into unconjugated and conjugated fractions which were further isolated and analysed by established methods. The injected hormone was rapidly metabolized with a half-life of approximately 10 min and metabolic clearance rate about 3.5 liters min. The bulk of the metabolites was found in the sulfate fraction from which a major metabolite was identified as 3 beta-hydroxy-5 alpha-pregnan-20-one. From the unconjugated fraction, 20 alpha-hydroxy-pregn-4-en-3-one, a known minor metabolite was also isolated. No radioactive estrogens were found. It is concluded that a major portion of circulating progesterone in the pregnant ewe near term is cleared by 5 alpha-reduction of ring A, followed by sulfo-conjugation.     

Leaf pretreatment with senescence retardants as a basis for oat protoplast improvement

Protoplasts obtained from oat leaves floated on buffer for 18hr show high nuclease activity, low rates of incorporation ofamino acids and nucleosides into macromolecules, and high ratesof spontaneous lysis. Addition to the leaf flotation mediumof the senescence retardants cycloheximide or kinetin, of thedibasic amino acids L-lysine or L-arginine, or of the diaminesputrescine or cadaverine reduces the rise in nuclease activityand spontaneous lysis of protoplasts, and increases the rateor extent of presumptive protein and nucleic acid synthesis.The diamines, which also retard chlorophyll degradation in theexcised leaves, appear to act both on the membrane and on systemscontrolling macromolecular synthesis and breakdown. By contrast,the senescence promoter L-serine hastens chlorophyll degradationfrom excised leaves and does not improve protoplasts derivedfrom those leaves. (Received July 4, 1977; )     

The Interactions of Superoxide Ion(O-2) with Metallo-Porphyrins [(C18TPP)M, M = FE,MN,CO,ZN]; Models for Biological Systems and Superoxide Dismutases

In dimethylformamide superoxide ion forms a l:l adduct with tctrakis (2.6-dichlorophenyl) porphinatoiron, (Cl₈ TPP)FeOO^-, as well as with its manganese analogue, (Cl₈ TPP)MnOO^-. On the basis of their electrochemical, spectroscopic, and magnetic properties these adducts have a metal-oxygen covalent bond (PorM-OO^-), oxygen-centered redox chemistry. and reactivities that are similar to the hydroperoxide ion (HOO^-). Addition of ^-OH to a solution of PorFe and O₂ results in the formation of PorFe(OH)(OO^-), which can be electrochemically oxidized to PorFeOH plus O₂ (-0.2 V vs SCE). Addition of protons to the PorM-OO^- adducts promotes their rapid decomposition to PorM, HOOH. and O₂. This chemistry provides insight to the reactions of biological superoxide and superoxide dismutases.     

Fine needle aspiration diagnosis of Penicillium marneffei infection

A disseminated infection with Penicillium marneffei, a rare human pathogen that may infect both healthy and immunocompromised patients, was diagnosed by fine needle aspiration cytology in a patient infected with the human immunodeficiency virus. The presence of yeast-form organisms with an eccentric or central dot and occasional septate and elongated forms highly suggested the diagnosis, which was confirmed on culture. Establishment of the diagnosis is important because this infection is potentially curable.