Intact form of myeloperoxidase from normal human neutrophils

Myeloperoxidase (donor: H₂O₂ oxidoreductase, EC 1.11.1.7) of human polymorphonuclear neutrophils was purified rapidly in the presence of the protease inhibitors phenylmethanesulfonyl fluoride and pepstatin A. The purified enzyme behaved as a single molecular species in several nondenaturing electrophoretic and chromatographic systems. Peroxidase activity in fresh extracts of neutrophils from 20 normal persons and from 5 patients with polycythemia was electrophoretically identical to purified enzyme. Treatment with trypsin converted myeloperoxidase to multiple electrophoretic forms of active enzyme. Size (M_r ca. 15,000 and ca. 55,000) and stoichiometry of the subunits of purified enzyme, and enzyme M_r ca. 140,000, were compatible with intact myeloperoxidase having an α₂β₂ structure. We found no evidence for electrophoretically detectable genetic polymorphism of myeloperoxidase. Proteolytic degradation of myeloperoxidase probably accounted for electrophoretic heterogeneity of enzyme and for some constituent peptides described previously.     

Sequential conversion of the glutathionyl side chain of slow reacting substance (SRS) to cysteinyl-glycine and cysteine in rat basophilic leukemia cells stimulated with A-23187

In rat basophilic leukemia (RBL-1) cells stimulated with A-23187, the major slow reacting substance (SRS) species contain glutathione, cysteinyl-glycine, or cysteine in their side chains, corresponding or closely related to leukotrienes LTC₄, LTD₄, and LTE₄, respectively.³ Evidence is presented that most of the SRS produced during the first few minutes of stimulation by the ionophore has a glutathionyl side chain which is sequentially converted to cysteinyl-glycine and cysteine.     

Evaluation of the role of calcitonin deficiency in ovariectomy-induced osteopenia

Studies were carried out in rats to examine the role of calcitonin deficiency in the pathogenesis of ovariectomy-induced osteopenia. The parathyroid glands of 80 female Wistar rats were autotransplanted to their thigh muscle and the animals divided into 4 groups. Group 1 rats were sham ovariectomized, and thyroidectomized to make them calcitonin deficient; Group 2 rats were thyroidectomized, and ovariectomized to make them deficient in ovarian hormones as well; Group 3 rats were sham thyroidectomized and sham ovariectomized, and Group 4 rats were sham thyroidectomized and ovariectomized. A fifth group of rats were unoperated upon and served as controls. Thyroidectomized animals were maintained on thyroxine replacement and 11 months after ovariectomy all the animals were bled, killed and their femurs dissected out. In both the thyroid intact and thyroidectomized animals, ovariectomy decreased femur density significantly (P less than 0.01). Similarly, ovariectomy resulted in a decrease in femur calcium (P less than 0.01) in both groups of animals, and in a significant decrease in serum calcitonin (P less than 0.05) in the thyroid intact animals. We conclude from these findings that ovarian hormone deficiency can cause bone loss independently of lowering circulating calcitonin levels.     

Localization of a site interacting with human platelet receptor on carboxy-terminal segment of human fibrinogen gamma chain

We report that the 27-residue carboxy-terminal cyanogen bromide fragment of human fibrinogen γ chain inhibits binding of [¹²⁵I]fibrinogen to human platelet receptors and blocks fibrinogen-mediated aggregation of ADP-treated human platelets. The blocking activity of the peptide was preserved after proteolysis of the isolated peptide with staphylococcal protease to generate a mixture of a dodecapeptide and a pentadecapeptide. Trypsin treatment destroyed blocking activity of the isolated peptide. These results indicate that the site responsible for the interaction of human fibrinogen with the platelet receptor resides in the 27-residue carboxy-terminal region of the γ chain.     

Contact sensitivity in rabbits sensitized with dinitrophenylated Mycobacterium tuberculosis

The conjugation of Mycobacterium tuberculosis with DNFB results in the formation of a haptenated preparation that induces the formation of contact sensitivity when administered subcutaneously. This contact sensitivity can be measured in vivo by topical application of the free chemical and in vitro by lymphocyte transformation. The antigens suitable for the in vitro detection are those preparations obtained by the haptenation of cell membranes. Haptenation of serum proteins, of homologous and heterologous origin, does not produce antigens suitable for in vitro assay. The antigen requirements for the in vitro transformation assay of contact sensitivity are similar for adjuvant induced sensitivity as well as for free chemical induced sensitivity.     

Incorporation of radiolabel from [1-14C]5-hydroperoxy-eicosatetraenoic acid into slow reacting substance

When synthetic [1-¹⁴C]5-hydroperoxy-eicosatetraenoic acid was incubated with rat basophilic cells, incorporation of the radiolabel into slow reacting substance (SRS) could be demonstrated as evidenced by comigration of spasmogenic activity and radioactivity after purification by high pressure liquid chromatography. This provides direct evidence that SRS is a product of the lipoxygenase pathway.     

Arylsulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is ome evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Transient lysozymuria during Sarcoma I tumor rejection

The present studies on the in vivo regulation of lysozyme synthesis by cytotoxic macrophages infiltrating Sarcoma I (Sa I) tumor ascites of C57BL/KS mice are an extension of previous observations that the elevation of serum and urinary lysozyme levels might reflect the degree of macrophage-mediated host resistance. Cytotoxic macrophages of host origin are required in the natural rejection of tumors by C57BL/Ks mice challenged with Sa I. The amounts of lysozyme in the serum and urine of the affected mice are quantitated during Sa I growth and rejection. The enzyme levels are correlated with the number of macrophages in the tumor and with the capacity for lysozyme synthesis measured in vitro on monolayers of macrophages isolated from the ascites. The results indicate that the elevation of serum and urinary lysozyme activity is a reflection of both the increased number of cytotoxic macrophages in the host and the qualitative change in the functional state of the cells. These parameters alone cannot account for the extent of the in vivo release of lysozyme in the serum and urine of the affected animals. High levels of lysozyme are present in the extracellular fluids of the mice only when the macrophages are actively participating in host defense. We conclude that in vivo control mechanisms which depend on the presence of viable tumor cells are responsible for the regulation of lysozyme release by cytotoxic macrophages. However, the high levels of lysozyme do not always indicate complete rejection, as indicated by a preliminary observation in which extensive lysozymuria was followed by tumor relapse.     

Formation of the cysteinyl form of slow reacting substance (leukotriene E4) in human plasma

The two major species of slow reacting substance (SRS) contain either a glutathionyl or cysteinyl-glycyl side chain. Incubation of these SRS's with undiluted or diluted (usually 1:10 or 1:50) human plasma at 37°C resulted in marked losses of smooth muscle contracting activity due primarily to conversion of their oligopeptide side chains to cysteine.     

Human Y-chromatin : III. The nucleolus

The relative positions of nucleoli and the Y-chromatin body were investigated in human interphase fibroblast nuclei to determine if the reported nucleolar association of the Y-body might be a chance phenomenon. Although nucleolar material was found to be mainly in the central area of the nucleus, the association of the Y-body with a nucleolus was highly significant, irrespective of the morphology or location of the Y-body within the nucleus. The association was corroborated with late interphase and early prophase nuclei in which nucleolar remnants were seen to concentrate around the Y chromosome.     

Relative contracting adn relaxing potencies of a series of prostaglandins on isolated canine mesenteric artery strips

The contracting and relaxing potencies of anf interactions between a number of prostaglandins (PGs) were studied $\text{in vitro}$ on spiral strips of small canine mesenteric arteries (outside diameter < mm). PGF₂α and PGE₂, the most potent contracting PGs, were nearly equal in potency (EC₅₀ 4 × 10^?7M) and did not cause relaxation under our experimental conditions. PGI₂ and PGE₁ were equal and the most potent relaxing PGs (EC₅₀ 3 × 10^?9M). PGE₁ also caused contraction, but this effect was not consistent. PGI₂ did not cause contraction in concentrations up to 3 × 10^?6M. In higher concentrations, however, it caused abrupt and near maximal contraction. PGD₂ was weak in both respect, causing incomplete relaxation and contraction or biphasic effects. Interaction studies showed that PGE₁ and PGI₂ mutually excluded the relaxing effects of each other. PGE₁ also reversed the relaxing effect of isoproterenol. However, pre-exposure to PGD₂ did not attenuate the relaxing effect of PGE₁ or PGI₂ nor was the relaxing effect of PGD₂ changed by pre-exposure to PGE₁. Two different orders of potency of PGs suggest two PG receptors subserving contraction and relaxation, respectively. Further, it appears that several PGs can act upon both receptors which may explain unusual interactions between the PGs and some of their atypical effects. Finally, the data also suggest that there may be subtypes of the PG receptors subserving contraction and relaxation.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured human fetal aortic smooth muscle cells.

Cultured human fetal aortic smooth muscle cells derived from the abdominal aorta converted benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) $\text{via}$ cytochrome P-450-dependent monooxygenation to metabolites detectable by both a highly sensitive radiometric assay and high pressure liquid chromatography (HPLC). Cells incubated with ³H-BaP transformed this substrate primarily to phenols. ¹⁴C-DMBA was converted to metabolites that cochromatographed with 12-hydroxymethyl-7-methylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz-[a]anthracene, 7,12-dihydroxymethylbenz[a]anthracene, and trans-8,9-dihydrodiol-7,12-DMBA. Exposure of cells in culture to 13 μM 1,2-benz[a]anthracene resulted in increased oxidative metabolism of both BaP and DMBA. In the case of BaP, total phenol formation was increased, while with DMBA all metabilities detected by HPLC were increased. Support for the potential role of metabolism of polycyclic aromatic hydrocarbons by aortic smooth muscle cells in the etiology of atherosclerosis was obtained.     

Selective 7-O-methylation of salsolinol in rat brain and heart in vivo

Intracerebroventricular and intraperitoneal injections of salsolinol and subsequent analyses of rat brain and heart revealed that 7-O-methylsalsolinol (7-OMe-Sal) is the principal O-methylated product formed in vivo. Production of 7-OMe-Sal was blocked by pretreatment of the rats with pyrogallol, an inhibitor of catechol-O-methyltransferase. 7-OMe-Sal and 6-O-methylsalsolinol (6-OMe-Sal) injected intraventricularly or prepared as external standards were distinguished by gas chromatography as peaks with different relative retention times compared to salsolinol as an internal standard. These substances were chromatographed as their corresponding pentafluoropropionic anhydride derivatives on a JXR column. Similar experiments performed with 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline revealed that this substance was similarly selectively converted to its 7-O-methylated product in rat brain.     

Comparison between electrically evoked and potassium-induced 3H-noradrenaline release from rat neocortex slices: Role of calcium ions and transmitter pools

In this study ³H-noradrenaline (NA) release from rat neocortex slices evoked by electrical field-stimulation (1 Hz, 12 mA, 2 msec) was compared with that induced by K⁺-depolarization (13–30 mM K⁺) under similar experimental conditions, with a particular emphasis on the role of external Ca²⁺ and the releasable transmitter pool(s). Not only ³H-NA release evoked by electrical stimulation but also that induced by 13 mM K⁺ was almost completely blocked by 0.3 μM tetrodotoxin (TTX). Release induced by 20 mM K⁺ appeared to be less sensitive to TTX. Thus, under relatively mild stimulation conditions, the activation of sodium channels appears to be involved in ³H-NA release elicited by both stimuli.The electrically evoked ³H-NA release increased sigmoidally with the external Ca²⁺-concentration up to 1.2 mM. In contrast, ³H-NA release induced by 13–20 mM K⁺ reached a maximal value at 0.6–0.9 mM Ca²⁺ and gradually decreased at higher Ca²⁺-concentrations. The Ca²⁺-antagonist D-600 (1–30 μM) did not inhibit electrically evoked release, while K⁺-induced ³H-NA release was dose-dependently reduced. Upon repetitive K⁺-depolarization a strong depression of ³H-NA release could be demonstrated, while this phenomenon did not occur with repeated electrical stimulation. Moreover, a previous K⁺-induced (partial) depletion of ³H-NA stores did not affect the release evoked by electrical pulses and vice versa. Taken together these data are compatible with a much stronger activation of Ca²⁺-channels and a larger vesicle mobilizing capacity in case of electrical stimulation at physiological frequencies compared to sustained depolarization with moderate K⁺-concentrations.     

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.     

Effects of alloxan-diabetes on the sodium-potassium adenosine triphosphatase enzyme system in dog hearts

The effects of alloxan-diabetes on the partial reaction of (Na⁺+K⁺)-ATPase, K⁺-activated para-nitrophenylphosphatase, and on ouabain binding were studied in isolated adult dog heart myocytes. The Km of K⁺-activated para-nitrophenylphosphatase for K⁺ activation was increased from 2.5 to 7.7 mM with no change in Vmax. The Scatchard plots for ouabain binding between control and diabetic animals were indistinguishable. These results indicate that in acute diabetes induced by alloxan, the number of Na⁺-K⁺ pumping sites in the heart is not altered but the affinity of the system for K⁺ is decreased. It is suggested that the decrease in K⁺ affinity of the (Na⁺+K⁺)-ATPase enzyme system is at least in part responsible for the altered K⁺ homeostasis in the diabetic state.