Changes in the properties of adenylate cyclase from guinea pig lungs in tuberculosis

Changes in the properties of adenylate cyclase from the lungs of tuberculotic guinea pigs were revealed. The number of beta-adrenergic receptors in the lungs was found to be reduced by 30% at the second and by 70% at the third stage of the disease. The degree and the value of Ka for adenylate cyclase activation by isoproterenol remained thereby unchanged. The basal activity of adenylate cyclase was increased by 20% against the control level at the second stage and decreased by 20% at the third stage of the disease. At these periods, the stimulating effects of guanylyl imidodiphosphate, NaF and forskolin on lung adenylate cyclase were diminished. The experimental results point to the significant role of the enzymes of cAMP metabolism and reflect the course of the tuberculosis process in experimental animals.     

Analysis of three-dimensional ciliary beating by means of high-speed stereomicroscopy.

Results are presented on the analysis of three-dimensional motion of compound cilia or cirri in voltage-clamped specimens of the protozoan Stylonychia mytilus. Time series of three-dimensional data were obtained by using the anaxial illumination method for simultaneous recording of stereoscopic video images. Data processing involved the following steps: determination of a reference coordinate system based solely on features present in each stereo-pair; tracing of cirral axes in digitized images, conversion to parameter curves by means of least-squares polynomial approximation, conversion of pairs of two-dimensional data to a series of three-dimensional data; correction for distortion due to projective shortening and conversion to a series of polynomial triplets, and analysis of the periodical components of the motion pattern in the frequency domain. Reconstructed beating cycles show typical differences between hyperpolarization-induced ciliary activity and depolarization-induced ciliary activity. Reconstructions of the motion of the basal segment of a cirrus are in agreement with existing data. Analysis of the curvature and torsion of a cirral axis during beating does not reveal any simple pattern of propagated activity within the axoneme. The return stroke may be subdivided into two phases. First, a curvature peak develops proximally. Secondly, a region with increased torsion arises more distally and spreads out in proximal direction. Both curvature and torsion return to minimal values by the beginning of the power stroke.     

Purification of smooth-muscle myosin free of calmodulin and myosin light-chain kinase. Susceptibility to oxidation.

Smooth-muscle myosin purified as described by Persechini & Hartshorne [(1983) Biochemistry 22, 470-476] contains trace amounts of calmodulin and myosin light-chain kinase, which can be removed by Ca2+-dependent hydrophobic-interaction chromatography followed by calmodulin-Sepharose affinity chromatography. The resultant column-purified myosin exhibits properties similar to those of the non-purified myosin, e.g. actin activation of the Mg2+-ATPase requires Ca2+/calmodulin-dependent phosphorylation of the two 20 kDa light chains. However, unlike the non-purified myosin, the column-purified myosin undergoes a time-dependent transition to a form which no longer requires phosphorylation for actin activation of the myosin Mg2+-ATPase. This transition is identified as a time-dependent change in conformation of the column-purified myosin from a 10 S to 6 S form and is caused by slow oxidation of the column-purified myosin, since it could be prevented by storage under N2 and reversed by 5 mM-dithiothreitol.     

Fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases. Application to subtilisins.

A fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases is described. It consists of: selective labelling of the corresponding subsites with a fluorescent group by using N alpha-dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ated peptide chloromethanes containing different numbers of amino acid residues, and probing the immediate environment of the subsites by quenching experiments using ionic and neutral quenchers. Intramolecular distances between the subsites and particular chromophores can be also determined. The technique is of general applicability to all serine proteinases. The above mentioned approach was applied to two proteinases: subtilisin Novo and mesentericopeptidase. It was concluded that the substrate-binding site of mesentericopeptidase is considerably more polar than that of subtilisin Novo. Intramolecular distances between the labelled subsites and tryptophan residues in the two proteinases were determined.     

Anti-My-26: a monoclonal antibody inhibiting human phagocyte chemiluminescence

Anti-My-26, a mouse monoclonal IgG1 antibody, was raised against human granulocytes and has been shown to inhibit luminol-enhanced, glucose-independent chemiluminescence (CL) of human granulocytes (or monocytes) responding to the soluble secretagogues A23187 or ionomycin (calcium ionophores) and phorbol myristate acetate (PMA). Anti-My-26 inhibition of CL was reversible and was dependent on both secretatogue and monoclonal antibody concentration. This inhibition appeared to be directed at the component of granulocyte CL that is independent of NAD(P)H-oxidase-catalyzed formation of superoxide anion, because neither opsonized zymosan-stimulated CL nor the PMA-induced decrease in NAD (P)H-associated autofluorescence was affected by anti-My-26. In addition, ionomycin, over a wide concentration range, failed to generate any decrease in granulocyte autofluorescence. The A23187-induced CL inhibited by anti-My-26 was correlated with its depression of oxygen consumption. Furthermore, anti-My-26 was not cytotoxic and did not itself induce oxidative metabolism when used as a stimulant. Binding of anti-My-26 to phagocytic cells was not decreased by pre-exposure of cells to either A23187 or PMA. Evidence is presented to suggest that the binding of anti-My-26 to the granulocyte surface inhibits the oxidative response to calcium ionophore and PMA by blocking a common pathway(s) stimulated by these different secretagogues.     

Influence of chronic antigen exposure on the metabolism of PGH2 by microsomal preparations of airway and parenchymal tissues in the sheep

The effects of repeated antigen exposure on the synthesis of mediators by lung tissues are not well understood. To investigate the influence of antigen challenge on the synthesis of prostaglandins by central airway and peripheral lung tissues, fourteen sensitive sheep underwent biweekly exposure to aerosolized $\text{Ascaris suu}$ antigen (7) or saline (7). Following the fifth exposure, microsomal and high speed supernatant fractions were prepared from trachealis muscle and lung parenchyma. Synthesis of thromboxane (TX) A₂, prostaglandin (PG) D₂ and PGI₂ from the PG endoperoxide intermediate, PGH₂, was assayed over a range of substrate concentrations from 3–200 uM. Synthesis of PGI₂ by trachealis microsomes was approximately 5-fold greater than that of TXA₂. PGI₂ and TXA₂ production was identical in tracheal preparations from Ascaris- and saline-exposed animals. In parenchymal tissues, where TXA₂ production predominated over PGI₂ by 9-fold, preparations from Ascaris- exposed animals synthesized 50% more TXA₂ than controls at PGH₂ concentrations of 25 uM and above, whereas synthesis of PGI₂ and PGD₂ were similar in preparations from both groups of animals. The density of pulmonary mast cells was decreased by 21% in the Ascaris group, whereas polymorphonuclear leukocyte density was unchanged. These results demonstrate the differential synthesis of TXA₂ and PGI₂ in central airways and peripheral lung regions of the sheep. They further indicate that repeated exposure of the airways to antigen selectively enhances TXA₂ synthesis in the lung periphery of sensitized animals. The site of this increased enzymatic activity, whether in resident cells or newly-infiltrated cells, has not been determined.     

Mechanism and dynamics of conformational ordering in xanthan polysaccharide

The thermally induced order-disorder transition of xanthan (extracellular bacterial polysaccharide from Xanthomonas campestris) has been investigated by optical rotation, differential scanning calorimetry, stopped-flow reaction kinetics and low-angle laser light scattering, and the results have been analysed in terms of Zimm -Bragg helix-coil transition theory. The reciprocal of the transition midpoint temperature (Tm) varies linearly with the logarithm of cation (K+) the salt dependence of Tm, is in agreement with Manning polyelectrolyte theory the ordered structure. The associated increase in cation binding, calculated from the salt dependence of tm, is in agreement with the Manning polyelectrolyte theory for one of the candidate structures from X-ray diffraction, a 5(1) single helix stabilized by packing of side-chains along the polymer backbone, but not for the alternative double-helix structure that has also been proposed. At each salt concentration, the two fundamental parameters of the Zimm -Bragg theory, s and sigma, were calculated. The equilibrium constant for growth of the ordered structure (s) is derived directly from calorimetric measurement of transition enthalpy (delta Hcal ), and sigma, which quantifies the relative instability of the helix nucleus, is derived from the ratio of delta Hcal to the apparent transition enthalpy (delta Happ ) obtained by van't Hoff analysis of the optical rotation data. The temperature course of conformational ordering calculated theoretically is in good quantitative agreement with experimental results from both optical rotation and scanning calorimetry. The calculated average length of stable, ordered chain-sequences increases with decreasing temperature, but equals or exceeds the total chain length from light scattering only at temperatures more than approximately equal to 70 K below Tm, suggesting that ordered and disordered regions may co-exist within the same xanthan molecule. Consistent with this interpretation, the observed rate of conformational ordering increases sharply under conditions where the starting solution for dynamic measurements is partially ordered, suggesting that ordered sequences within each chain may act as helix nuclei for adjacent disordered regions, so that helix growth, rather than the slower nucleation process, becomes rate limiting.