Metabolism of prostaglandin endoperoxide by microsomes from human lung parenchyma and comparison with metabolites produced by pig, bovine, rat, mouse and guinea-pig

The metabolism of PGH2 by human lung parenchymal microsomes was characterized by radiometric high performance liquid chromatography and compared with metabolism by pig, bovine, rat, mouse, and guinea pig lung microsomes. Microsomes from human lung synthesized 0.74 nmoles/mg protein and 0.72 nmoles/mg protein, PGI2 (6-Keto-PGF1 alpha) and TxA2 (TxB2) respectively, upon incubation with 4.0 nmoles of PGH2. Pig, bovine, rat, mouse, and guinea pig microsomes respectively synthesized 1.0, 1.0, 0.9, 0.4, and 0.1 nmoles of PGI2/mg protein, and 0.9, 1.0, 0.7, 0.3, 1.8 nmoles of TxA2/mg protein, and preparations formed some PGE2, PGF2 alpha, and PGD2. Mouse lung microsomes were unique in synthesizing PGE2 as the major prostaglandin. The thromboxane synthetase inhibitor 1-benzylimidazole was a specific inhibitor in these six species.     

Human duodenal gland (Brunner's gland) mucus glycoprotein analysis

A mucus glycoprotein of the duodenal gland is characterized. The glycoprotein was isolated from a water-soluble homogenate fraction of the submucosal tissue of the most proximal part of the small intestine, containing the duodenal gland, and was purified from contaminating protein by two sequential equilibrium-centrifugation steps in CsCl density gradients. Structural analysis of the purified glycoprotein showed two regions in the protein core: one part characterized by the presence of essentially all of the cysteine residues and another by the presence of most of the serine and threonine. Carbohydrate was found linked to the latter part. Rat (H. L. Smits, P. J. M. van Kerkhof, and M. F. Kramer (1982) Biochem. J. 203, 779-785.) and human duodenal gland mucus glycoprotein show homology in chemical composition. Both glycoproteins have a relatively high protein content and contain little sulfate and no neuraminic acid. In man the mucus glycoprotein, however, has a higher content of serine plus threonine, a lower content of N-acetylglucosamine, a slightly higher content of fucose, and a lower molar ratio of N-acetylgalactosamine relative to serine plus threonine.     

12-O-tetradecanoylphorbol-13-acetate stimulates release of arachidonic acid, prostaglandin E2 and prostaglandin F2 alpha from TPA nonproliferative variants of 3T3 cells

The Proteinase Inhibitor Inducing Factor, PIIF, a pectic polysaccharide that induces synthesis and accumulation of proteinase inhibitor proteins in tomato and potato leaves, is an effective elicitor of the phytoalexin pisatin in pea pod tissues. The levels of pisatin induced by PIIF, and the time course of elicitation, are similar to those induced by chitosans, β-1,4 glucosamine polymers, which are potent elicitors of pisatin in pea pods. Similarly, the chitosans, found in both insect and fungal cell walls, are the most potent inducers yet found of proteinase inhibitor accumulation in excised tomato cotyledons. The similarity in the induction of synthesis of proteinase inhibitors in tomato cotyledons and of pisatin in pea pods by pectic polysaccharides and chitosans suggests that the two polysaccharide types may be triggering a similar fundamental system present in pea and tomato plants that regulates the expression of genes for natural protection systems.     

Changes of gamma-glutamyltranspeptidase activity in the rat during development and comparison of the fetal liver, placental and adult liver enzymes

Changes in the activity of gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) during development in rats were investigated. The activity of GGT in fetal liver increased rapidly immediately before birth, reached a maximum at birth and then decreased rapidly within a week after birth to nearly the level in adult rat liver. In contrast, placental GGT showed higher activity at an early stage (from day 13 to day 15) of the gestation period, but the activity decreased in the last part of fetal life. The activity in the amniotic fluid increased significantly just before birth, in parallel with the increase of activity in the fetal liver. No change of activity in the uterine wall was observed throughout gestation. The kinetic and immunological properties of partially purified GGTs from fetal liver and placenta were almost identical with those of adult liver GGT. However, the activity of soluble GGT in fetal liver was less effectively inhibited by antibody against adult kidney GGT. Thus, it is likely that at least one isozyme of GGT is present in the soluble fraction of fetal liver.     

Effects of a teratogen on [3H]uridine nucleotide transfer between human embryonal cells and on gap junctions

Intercellular communication (IC) mediated by gap junctions (gj) occurs during embryonal development and appears to be important for normal differentiation through the exchange of morphogenetic signalling substances. Disruption of IC by chemicals may induce abnormal development resulting from failed cell-cell interactions. It was established in the present study that genotypically normal human embryonal palate mesenchyme (HEPM) cells displayed IC in cell culture and that the transfer of [3H]uridine nucleotides was inhibited by the potent embryotoxin and teratogen 12-O-tetradecanoyl-phorbol-13-acetate (TPA). IC was mediated by gj which were revealed by freeze-fracture and electron microscopy. Quantitative morphometric analysis showed that inhibition of IC by TPA coincided with a significant reduction in the number of gj. The observations suggest that inhibition of IC by the teratogen TPA may be one among the many mechanisms believed to be responsible for the induction of abnormal development by chemical teratogens.     

The production and storage of Nerve Growth Factor in vivo by tissues of the mouse,rat, guinea pig,hamster, and gerbil

The Nerve Growth Factor (NGF) content in vivo of tissues from the mouse and rat at various stages of development from 3 days embryonic gestation to the attainment of full maturity has been determined using the standard biological assay. A less extensive survey has also been made of tissues from the guinea pig, hamster, and gerbil. With the exception of the well-documented high levels of NGF in the mouse submaxillary glands, none of the organs examined contained detectable NGF. These results, which are consistent with those previously reported using the biological assay, stand in contrast to the high levels of NGF detected in virtually all tissues by some published radioimmunoassays. It is likely that the discrepancies are due to the use in the radioimmunoassays of antisera containing antibodies to proteins other than NGF, and to the inability of one-site radioimmunoassays to distinguish between the presence of NGF and that of agents capable of binding NGF. The apparent lack of widespread NGF production in vivo contrasts with the ability of many tissues to synthesize the protein in vitro. This may imply that physiologically significant levels of NGF are below the limits of sensitivity of the assay systems presently available, that NGF synthesis in vivo occurs only during a very restricted period of development, or that the presence of a normal innervation pattern influences NGF production.     

Cellular aging studied by the reconstruction of replicating cells from nuclei and cytoplasms isolated from normal human diploid cells

Cytochalasin B (CB) was used to enucleate cells (cytoplasts) and to obtain karyoplasts (nuclei) from the human diploid fetal lung fibroblast strain WI-38. Fusion of cytoplasts and nuclei from young and old cells was accomplished with the aid of inactivated Sendai virus. Viable nuclei may be obtained from the karyoplast pellet after passage through a layer of bovine albumin which retains any contamination cytoplasts. The majority of successful fusions forming “whole cells” occurred when cytoplast from “old” cultures (PDL 40–51) and karyoplasts from “young” cultures were used (PDL 12–22), but almost always resulted in limited division of the viable reconstructed cells. When successful fusion occurred between “young” cytoplasts and “young” karyoplasts the number of cell divisions obtained was comparable to control cells kept under similar conditions.     

Metabolism of furazolidone by milk xanthine oxidase and rat liver 9000g supernatant: formation of a unique nitrofuran metabolite and an aminofuran derivative

In vitro metabolism of furazolidone (N-(5-nitro-2-furfuryliden)-3-amino-2-oxazolidone) was investigated by using milk xanthine oxidase and rat liver 9000g supernatant. As a result, a new type of reduction product was isolated as one of the main metabolites from the incubation mixture and it was tentatively identified as 2,3-dihydro-3-cyanomethyl-2-hydroxyl-5-nitro-1a, 2-di(2-oxo-oxazolidin-3-yl)iminomethyl-furo[2,3- b]furan. In addition, the present study demonstrated the formation of N-(5-amino-2-furfurylidene)-3-amino-2-oxazolidone as a minor metabolite of nitrofuran in a milk xanthine oxidase system. The aminofuran derivative was easily degraded by milk xanthine oxidase under aerobic, but not anaerobic, conditions. The degradation appears to be due to superoxide anion radicals, hydroxyl radicals, and/or singlet oxygen, which are produced in this enzyme system.     

Stimulation of myometrial and decidual prostaglandin production by amniotic fluid from term,but not midtrimester pregnancies

The effect of amniotic fluid obtained from second trimester (16–20 wks) and term pregnancies (38–41 wks) on the production of PGE and F by human amnion, decidua and myometrium at term was determined using tissue slices incubated in vitro. Midpregnancy amniotic fluid neither inhibited nor stimulated the prostanoid production by any of the tissues. In contrast, term amniotic fluid obtained before as well as after the onset of labor markedly increased the production of both PGE and PGF in decidua and myometrium from levels in Krebs solution. The prostanoid production (PGE + PGF) in amnoin was not significantly increased but the proportion of PGF was raised during incubations in term amniotic fluid. In decidua and myometrium the increase in PGE and PGF production in term amniotic fluid was approximately 200 and 400 percent respectively, from control values in Krebs solution. We propose that the stimulatory activity in term amniotic fluid in responsible for the accelerated synthesis of prostaglandins after of membranes, which is reflected in raised PGF metabolite levels in maternal circulation. It may also be the reason for the rise in amniotic fluid prostaglandin levels around the 35th week of gestation, and perhaps for the onset of labor.     

Characterization of a lymphokine produced by human T cells which inhibits collagen synthesis

Human lymphocytes, isolated from peripheral blood, were cultured for 48 hr in a defined medium containing 10 mg/ml bovine serum albumin and phytohemagglutinin. A lymphokine which inhibits collagen synthesis by cultured human dermal fibroblasts was purified from the lymphocyte incubation medium by successive steps of ammonium sulfate precipitation, gel filtration chromatography, and isoelectric focusing. Good recovery of this collagen synthesis inhibitory factor (CSIF) was obtained and a factor with an approximate molecular weight of 55,000 and a pI of 6.2 was isolated. The purification of the factor should permit further studies on its mechanism of action.     

Structure of lipoprotein (a) studied by spin-labeling

The potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate causes a 2-fold increase in 1,2-diacylglycerol levels within 15–30 min in cultured chick embryo differentiated myoblasts. The weak tumor promoter 12-O-decanoylphorbol 13-acetate was 250 times less effective and the non-promoter 4-α-phorbol 12,13 didecanoate was ineffective at producing this response. During subcellular fractionation, the stimulated portion of the diacylglycerol was distributed similarly to the plasma membrane fraction. Evidence is presented that this diacylglycerol originates from pre-existing lipid rather than from $\text{de}\text{novo}$ synthesis. Possible implications of these findings with regard to the inhibition of myoblast fusion by the tumor promoter are discussed.     

Radioimmunoassay for pig angiotensin I converting enzyme: a comparison of immunologic with enzymatic activity.

Angiotensin I converting enzyme in body fluids and extracts of various pig tissues was measured by radioimmunoassay and by enzymic assay. Based on the ratios of enzymic to immunologic activity, the extracts could be separated into two groups. One group, with ratios around 4 U/mg, included urine and extracts from the adrenal, choroid plexus, epididymis, gall bladder, heart, liver, retina, spleen, and testis. The other group, with ratios around 12 U/mg, contained serum and extracts from lung and kidney. Explanations are offered for why one group had a lower enzymic to immunologic ratio than the other.     

Correlation of biochemical and morphological changes induced by chemical injury to the lung

Comparison has been made of injury to the rat pulmonary alveolar parenchyma evoked by intravenous injection of N-nitrosomethylurethane, intratracheal instillation of 3-methylcholanthrene or repeated inhalation for up to 15 days of carbon tetrachloride, trichloroethylene or gasoline vapour. Biochemical analyses, including assessment of rates of RNA and DNA synthesis and secretion of pulmonary surfactant, were correlated with morphological changes determined by electron microscopy. Single doses of N-nitrosomethylurethane or 3-methylcholanthrene inhibited incorporation of [14C] orotate into lung RNA 1--3 days after treatment. Daily exposure for 30 min to carbon tetrachloride or trichloroethylene vapour caused less marked reduction in orotate incorporation. Ultrastructural examination revealed that 3-methylcholanthrene toxicity was characterised by cytoplasmic change including disruption of surfactant lamellaie of Type 2 pneumocytes and variable degenerative changes Type 1 pneumocytes. Eight to ten days after treatment, the morphological evidence of hypertrophy/hyperplasia and transformation of Type 2 pneumocytes correlated well with biochemical evidence of stimulated incorporation of [3H]thymidine. Inhalation of carbon tetrachloride or trichloroethylene vapour produced milder responses including occasional degenerative changes in Type 1 pneumocytes, reduced numbers of surfactant lamellae in Type 2 pneumocytes and no change in [3H]thymidine incorporation. In contrast to the gradation of injury produced by the various chemicals, all procedures caused a marked and reproducible reduction in secretion of pulmonary surfactant as determined by endobronchial lavage. Following solvent inhalation, reduced recovery of surfactant was detected within 5 days of repeated exposure and thereafter no further change in this depressed level resulted from continued exposure for a further 10 days. The data are discussed in terms of a generalised pattern of response by pulmonary alveolar tissue to chemical injury and the apparent sensitivity of surfactant secretion as an indicator of damage to the lung.     

Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies

We have identified three germ cell surface carbohydrate antigens that exhibit a common, stage-specific pattern of expression during spermatogenesis in the mouse. IgM-class monoclonal antibodies designated "J1," "C6," and "A5" were absorbed by adult testis, but not by any adult somatic tissue tested. In indirect immunofluorescence assays using collagenase-dissociated prepuberal and adult testicular cells, these antibodies labeled the surfaces of early and late pachytene spermatocytes and round spermatids. Gonocytes from fetal and neonatal testes were not labeled. In paraffin sections of prepuberal and adult testes, sialidase treatment exposed antigens recognized by antibodies C6 and A5 on preleptotene, leptotene, and zygotene spermatocytes located near the perimeter of seminiferous tubules. The determinants recognized by antibodies J1, C6, and A5 were characterized partially using a sugar hapten inhibition assay. The binding of J1 to adult testicular cells was inhibited specifically by N-acetylglucosamine and the binding of both C6 and A5 was inhibited by N-acetyllactosamine. The glycoconjugates recognized by J1, C6, and A5 eluted from gel filtration columns with an apparent molecular weight greater than 1 X 10(6) and were sensitive to endo-beta-galactosidase (keratanase) treatment. The apparent high molecular weight of these glycoconjugates was confirmed by immunolabeling Western blots of testis extracts separated by SDS-polyacrylamide gel electrophoresis. The results suggest that polylactosamine (keratan) glycoconjugates of high molecular weight are associated with the plasma membranes of meiotic and haploid male germ cells. The effects of sialidase on antibody labeling patterns suggest that changes in cell surface sialylation accompany the transition of early meiotic germ cells to pachytene spermatocytes during spermatogenesis.     

Demonstration of aromatic l-amino acid decarboxylase activity in human brain with l-dopa and l-5-hydroxytryptophan as substrates by high-performance liquid chromatography with electrochemical detection

The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. K_m and V_max values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 μM and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 μM and 71 pmol/min/g wet weight, respectively.     

Specific binding and pharmacological interactions of apamin,the neurotoxin from bee venom,with guinea pig colon

This paper describes the interaction of apamin, the bee venom neurotoxin, with its receptor in the guinea pig colon. The pharmacological activity of the toxin was assayed by measuring its contracting effect on guinea pig colon preparations that had been previously relaxed by neurotensin. The IC₅₀ value of apamin in this $\text{in vitro}$ bioassay is 7 nM. These pharmacological data are compared to the binding properties of apamin to smooth muscle membranes prepared from guinea pig colon. The highly radiolabeled monoiododerivative of apamin binds to its colon receptor with a dissociation constant $\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 36}\text{pM}$. The maximal binding capacity of colonic membranes is 30^dfmol/mg of protein. The dissociation constant of the unmodified toxin is 23 pM. The difference between the toxin concentrations that produce half-maximal effects in the binding and pharmacological studies arises from the different experimental conditions used for the two assays.     

Oxygen-derived radicals from Trypanosoma cruzi-stimulated human neutrophils

This study provides biochemical and electron spin resonance spectroscopic evidence that contract of human polymorphonuclear leukocytes with antibody-coated Trypanosoma cruzi triggers the respiratory burst. Oxygen consumption, superoxide anion and hydrogen peroxide release were stimulated under conditions of polymorphonuclear leukocyte-mediated killing. This stimulation did not occur under non-killing conditions when antibody was omitted. A common mechanism of cytotoxicity of human polymorphonuclear leukocytes against different T. cruzi forms is suggested by the triggering of the respiratory burst by antibody-coated epimastigotes and trypomastigotes.     

Identification of immunogens of Mycoplasmapneumoniae by protein blotting

Proteins of $\text{Mycoplasma}\text{pneumoniae}$ were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose sheet by blotting. Sera obtained from infected hamsters and immunized rabbits were then incubated with the nitrocellulose strips. Proteins which are capable of eliciting antibodies were detected by indirect immunoradioautography using ¹²⁵I-labeled antisera against hamster or rabbit IgG. Antibodies to seven immunogens were demonstrated in the sera of hamsters infected with $\text{M.}\text{pneumoniae}$ by inhalation, while many more proteins were found to be capable of stimulating antibodies in rabbits immunized parenterally with mycoplasmas. This method should prove useful for identifying those components of a microorganism which elicit antibody responses and contribute to the protective state.     

Cathepsin D generates gamma-endorphin from beta-endorphin.

A crude extract of porcine anterior pituitary was found to contain endopeptidase activity that splits the Leu⁷⁷-Phe⁷⁸ peptide bond of β-lipotropin in a pH range 3.0–7.0. The specificity and susceptibility to pepstatin of pituitary extract were the same as those of cathepsin D (E.C. 3.4.23.5) isolated from calf brain by affinity chromatography. Cathepsin D was shown to split the same Leu-Phe peptide bond of β-endorphin, leading to the formation of γ-endorphin. Based on the above data it is suggested that cathepsin D is a major factor in the generation of endorphins of intermediate size.     

Some characteristics of 75Se-P,a selenoprotein found in rat liver and plasma,and comparison of it with selenoglutathione peroxidase

The selenoenzyme glutathione peroxidase cannot account for all the physiological effects of selenium in rat liver. Therefore, a study was carried out with the ultimate aim of identifying selenoproteins other than glutathione peroxidase. The incorporation of ⁷⁵Se, given as ⁷⁵SeO₃^2?, into centrifugally separated fractions of selenium-deficient and control rat livers was determined. In selenium-deficient liver much less ⁷⁵Se was incorporated into the 105,000g supernatant fraction than in controls, so this fraction was studied further by gel filtration, ion-exchange, and hydroxylapatite chromatography. Selenoglutathione peroxidase and another selenoprotein, called ⁷⁵Se-P, were separated and identified. Both these selenoproteins were also found in plasma. Selenium deficiency had opposite effects on incorporation of ⁷⁵Se by these proteins. It decreased ⁷⁵Se incorporation by glutathione peroxidase at 3 and 72 h after ⁷⁵Se injection but increased ⁷⁵Se incorporation by ⁷⁵Se-P. This suggests that ⁷⁵Se-P competes for available selenium better than does glutathione peroxidase when the element is in short supply. Apparent molecular weights of ⁷⁵Se-P from liver and plasma determined by gel filtration were, respectively, 83,000 and 79,000, which indicate proteins smaller than glutathione peroxidase. Cycloheximide pretreatment of the rat blocked ⁷⁵Se incorporation into plasma ⁷⁵Se-P. These experiments establish the existence of a selenoprotein, ⁷⁵Se-P, in rat liver and plasma which is chromatographically distinct from glutathione peroxidase and which incorporates ⁷⁵Se differently from glutathione peroxidase. ⁷⁵Se-P may account for some of the physiological effects of selenium.