Actin Enables the Antimicrobial Action of LL-37 Peptide in the Presence of Microbial Proteases

Host defense peptides play an important host-protective role by their microcidal action, immunomodulatory functions, and tissue repair activities. Proteolysis is a common strategy of pathogens used to neutralize host defense peptides. Here, we show that actin, the most abundant structural protein in eukaryotes, binds the LL-37 host defense peptide, protects it from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis, and enables its antimicrobial activity despite the presence of the proteases. Co-localization of LL-37 with extracellular actin was observed in necrotized regions of samples from oral lesions. Competition assays, cross-linking experiments, limited proteolysis, and mass spectrometry revealed that LL-37 binds by specific hydrophobic interactions to the His-40–Lys-50 segment of actin, located in the DNase I binding loop. The integrity of the binding site of both LL-37 and actin is a prerequisite to the binding. Our results demonstrate that actin, presumably released by dead cells and abundant in infected sites, might be utilized by the immune system to enhance spatio-temporal immunity in an attempt to arrest infection and control inflammation.     

Insulin-like growth factors in sheep thyroid cells: action, receptors and production

Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 micrograms of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions, [125I]IGF-I bound to a moiety of approximate Mr = 135,000 and [125I]IGF-II to a moiety of approximate Mr = 260,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr = 34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner.     

Über einige Probleme der hypothalamischen Neurosekretion

Zusammenfassung Bei weißen Ratten hatte die Untersuchung der Nervenzellen des vorderen Hypothalamus in den verschiedenen Funktionsphasen und mit verschiedenen histochemischen Methoden zu der Folgerung Anlaß gegeben, daß die aktivierte Ganglienzelle zu Beginn mit großer Intensität Gömöri-positiven Stoff bildet, der später mobilisiert wird. Denn bei den Tieren, die nach starken akuten Einwirkungen bzw. nach der folgenden Hyperfunktion der Zwischenhirnkerne getötet wurden, lassen sich in lebhafter Sekretion befindliche Zellen und solche hypertrophische Formen, deren Sekret schon mobilisiert wurde, gleicherweise beobachten. Nach intensiven langdauernden Einwirkungen sieht man aber zumeist die zweiterwähnte, entleerte, eine ausgesprochene perinucleäre Aufhellung zeigende Form. Weiterhin hat es den Anschein, daß die hypothalamischen Nervenzellen keine Speicherfunktion haben: Sind sie mit Sekret gefüllt, handelt es sich wahrscheinlich um die Erzeugung des Sekretes. Für diese Annahme spricht auch die biphasische Restitution dieser Hirnkerne; in der ersten Phase sieht man dort viele sekretgefüllte Zellen, in denen das Produkt nicht gespeichert wird, sondern bald auf das Kontrollniveau fällt und sich in der Neurohypophyse ansammelt.Auf Grund der Änderungen der Menge des basophilen Stoffes der Ganglienzelle (Ribonucleinsäure, Nissl-Körnchen) und der Gömöri-Substanz sowie der Größenänderungen der Zellbestandteile unterscheidet Verfasser nachstehende, morphologisch verfolgbare Funktionsabschnitte in den Zwischenhirnkernen der weißen Ratte: Normalzustand, beginnende Hyperfunktion, verzögerte Hyperfunktion, erste Phase der Restauration, zweite Phase der Restauration (echte Normalisierung).Schließlich wird die Theorie Neurosekret als Produkt einer physiologischen Degeneration erörtert.     

Induction of ornithine decarboxylase activity by growth and differentiation factors in FRTL-5 cells

Induction of ornithine decarboxylase has been correlated with the onset of cellular proliferation and cAMP production. Whether the resulting increases in polyamine levels are essential mediators of growth and/or differentiation or are merely incidental remains controversial. We have used FRTL-5 thyroid cells in culture to study the effects of three growth factors on ornithine decarboxylase activity. These factors [TSH, bovine calf serum, and 12-O-tetradecanoylphorbol-13-acetate (TPA)] are thought to act through different intracellular pathways. TSH stimulates cAMP production in thyroid cells, calf serum acts through ill-defined pathways to stimulate growth, and TPA is known to activate protein kinase C. Bovine calf serum and TSH acted synergistically to induce ornithine decarboxylase activity. Activity was maximal when the phosphodiesterase inhibitor, methyl isobutyl xanthine, was included. Individually, neither serum nor TSH was a potent stimulator of the enzyme. Ornithine decarboxylase mRNA was apparent on Northern blots as a doublet following one hour of exposure to these agents. TPA did not stimulate ornithine decarboxylase activity and had an inhibitory effect on enzyme induction by TSH and serum. Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, inhibited growth induced by both TPA and TSH in putrescine-free medium. This effect was not apparent in medium containing 10(-5) M putrescine. The data indicate that, although intracellular levels of cyclic AMP regulate ornithine decarboxylase activity, a component in serum is necessary for significant induction of this enzyme. Factors stimulating growth by non-cyclic AMP-dependent pathways may act without apparently stimulating this enzyme, although polyamines appear to be essential for their growth stimulatory effects.     

Effect of Oxidized Spermine and Other Aldehydes on the Infectivity of Vaccinia Virus

The antiviral activity of oxidized spermine was compared with that of other aldehydes. Suspensions of vaccinia virus were incubated at 37 C with various concentrations of the aldehydes, and the infectivity of the viruses was determined by the plaque assay. Oxidized spermine at a concentration of 0.82 mM completely inactivated a suspension of vaccinia virus (1.4 x 10(8) plaque-forming units) after incubation at 37 C for 10 hr. Glutaraldehyde and formaldehyde were less active when compared on a molar basis, but acrolein resembled oxidized spermine in its antiviral activity. Because acrolein is produced from oxidized spermine at only 20 to 30% yield, it is unlikely that the biological activity of the latter is due to acrolein formed during the spontaneous degradation of oxidized spermine.     

Practical Procedures for the Purification of Bacterial Viruses

The efficiencies of the various methods used for phage concentration have been compared. The two-phase concentration method (with polyethylene glycol and dextran sulfate) gave maximal recoveries of infectivity for coliphages of the T-even and T-odd series and for ribonucleic acid phages and single-stranded deoxyribonucleic acid phages. Precipitation of phages by acid gave high yields when applied to T2 and T4 phages but not with T3 and T7 coliphages. Differential centrifugation was efficient when sedimented phages were gently dispersed before repeating the centrifugation cycle. The efficiencies of the various methods have also been confirmed by electron microscope studies, which also show that the two-phase concentration method gave rise to intact phages. Zone centrifugations in sucrose gradients (12.5 to 52.5%) indicated that coliphages of the T-even series sediment faster than T-odd coliphages; they may thus be separated from each other and from empty ghosts by centrifugation at 100,000 x g for 40 min. Equilibrium centrifugation in preformed cesium chloride gradients was also useful for phage concentration and purification. This study also deals with some optical properties of purified phages; optical cross sections and absorbance ratios (at 260 and 280 nm) of the various preparations are given.     

Disparate uptake of 99mTcO4- and 125I- in thyroid cells in culture

Pertechnetate (99mTcO4-) is a large anion that is thought to be trapped but not organified by the thyroid. In order to determine its usefulness in discriminating trapping from organification in thyroid cell culture, the uptake of 99mTcO4- has been compared to that of 125I- both in sheep thyroid cells and in FRTL5 cells, a functional rat thyroid cell line. We found that uptake of both isotopes was dependent on TSH or agents that raised intracellular cAMP levels and was displaceable with iodide in both cell types. However in rat cell line 99mTcO4- uptake was up to eight-fold higher than 125I- uptake, and 99mTcO4- but not 125I- uptake was doubled by methimazole treatment. A considerable proportion of 99mTcO4- but not 125I- taken up by the FRTL5 cell but not the sheep cells was precipitable with TCA. This proportion was increased by methimazole treatment. We also found marked differences in sensitivity to ouabain between the two cell types. These results illustrate the differences between species in 125I- and 99mTcO4- transport and support the concerns expressed by Socolow and Ingbar (1967) in using 99mTcO4- as a direct and precise indicator of iodide transport activity.     

Polyamines and tumor cells: effect of transformation of chick embryo fibroblasts by Rous sarcoma virus on polyamine levels

The effect of transformation of chick embryo fibroblasts, by Rous sarcoma virus, on intracellular polyamine levels has been studied. A good correlation between spermidine and cellular protein content has been demonstrated. Upon changing the medium, a sharp increase in spermidine level was noticed both in normal and transformed cells. This increase was accompanied by enhanced protein synthesis. The intracellular concentrations of spermine and spermidine were very similar in normal and transformed cells. On the other hand, significant differences in putrescine levels were demonstrated: in normal cultures the intracellular concentration of putrescine reached a plateau approximately 6 days after seeding, whereas a continuous rise of the diamine in transformed cells was noticed. These differences, which were observed in cultured cells, may explain the known accumulation of polyamines during neoplastic growth.