Retention of topical liposomal formulations on the cornea

The ability of liposomes composed of different kinds of phospholipid materials to adhere to the surface of the cornea was studied in the rabbit. The liposomes were labelled with tracer amounts of an I125-labelled phosphatidylethanolamine derivative and were instilled in 10 microliters drops onto the cornea. The retention of radioactivity was monitored. The results show that liposomes containing positively charged phospholipids are better retained than an albumin control. Thus, it may be possible to develop a drug delivery liposome system which would permit long-term sustained release of ophthalmic drugs onto the cornea.     

Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes

Protein transduction domains (PTDs) are used to enhance cellular uptake of drugs, proteins, polynucleotides or liposomes. In this study, functionalized Antennapedia (Antp, aa 43–-58) and HIV Tat (aa 47–57) peptides were coupled to small unilamellar liposomes via thiol-maleimide linkage. Modified liposomes showed higher uptake into a panel of cell lines including tumor and dendritic cells than unmodified control liposomes. Liposome uptake was time and concentration dependent as analyzed by flow cytometry and live-cell microscopy. At least 100 PTD molecules per small unilamellar liposome (100 ± 30 nm) were necessary for efficient translocation into cells. Cellular uptake of PTD-modified liposomes was 15- to 25-fold increased compared to unmodified liposomes and was inhibited by preincubation of liposomes with heparin. Glycosaminoglycan-deficient CHO cells showed dramatically reduced cell association of PTD-modified liposomes, confirming the important role of heparan sulfate proteoglycans in PTD-mediated uptake. Antp-liposomes used as carriers of the cytotoxic drug N⁴-octadecyl-1--D-arabinofuranosylcytosine-(5- 5)-3-C-ethinylcytidine showed a reduction of the IC₅₀ by 70% on B16F1 melanoma cells compared with unmodified liposomes. PTD-functionalized liposomes, particularly Antp-liposomes, represent an interesting novel carrier system for enhanced cell-specific delivery of a large variety of liposome-entrapped molecules.Received 16 April 2004; received after revision 13 May 2004; accepted 25 May 2004     

An epoxidation byCorynebacterium simplex

Zusammenfassung Während unserer Untersuchungen über die mikrobiologische Dehydrierung verschiedener 16-Methylsteroide durch Bakterienkulturen vonCorynebacterium simplex, wurde eine neue Art von Umwandlung beobachtet. Von 16-Methyl-9(11)-pregnen-17, 21-diol-3, 20-dion-21-acetat ausgehend, gelang es aus den Kulturfiltraten 16-Methyl-1, 4-pregnadien-9, 11-epoxy-17, 21-diol-3, 20-dion zu isolieren. In einigen Fällen wurde auch das4-Derivat dieser Verbindung als Nebenprodukt erhalten.     

Induction of lymphokine-activated killer cells from nude mouse spleen cells by interleukin-4

The induction of lymphokine-activated killer (LAK) cells from natural killer (NK) lineage cells by interleukin-4 (IL-4) was studied in vitro. Activation of nude mouse spleen cells by IL-4 generated cytotoxic cells, capable of killing NK-sensitive as well as NK-resistant tumor cells. The induction of peak lytic activity was demonstrated after 3 days of culture with IL-4. Surface marker analysis indicated that the majority of precursor cells were aGM1⁺, Thy1^–, and the majority of effector cells were aGM1⁺, Thy1⁺, suggesting that IL-4 induced LAK cells from nude mouse spleen cells were similar to those from normal mouse spleen cells. The induction of nude mouse LAK cells by IL-4 was partially inhibited by anti-IL-4 or anti-interferon (IFN)-, antibody, and it was further inhibited by the combination of two antibodies, suggesting that IFN-, production was associated with LAK induction of NK lineage cells by IL-4.     

Deciduogenic effects of mediators of the polyphosphatidylinositol pathway in pseudopregnant mice

Intraluminal injections (15 l) of either concanavalin A (125 g) or ionophore A 23187 (0.01 mol) induced a decidual cell reaction (DCR) in the uterus of day 4.5 pseudopregnant mice. However, when these agents were administered in different combinations with each other or with CaCl₂ (15 mol) and phorbol-12-myristate-13-acetate (1.6 nmol), interacting effects occurred to either enhance or inhibit each of the others' independent deciduogenic capacities. The results suggest that the polyphosphatidylinositol pathway and Ca²⁺ are involved in the induction of the DCR in mice with complex interactions occurring between the active components of the pathway to modulate the outcome of the transformation process.     

Epidermal growth factor stimulates proliferation of rat hepatoma cells producing α-fetoprotein

Summary Epidermal growth factor stimulated both [³H]thymidine uptake and proliferation of rat AH66 hepatoma cells. However, the increase in cell number was not accompanied by a proportional increase in the levels of -fetoprotein of the culture media. The effects of EGF on the cell proliferation were antagonized by N⁶, O²-dibutyryl cAMP.     

Evidence for the extranuclear localization of thymosins in thymus

A new radioimmunoassay has been developed for thymosin ₄ by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1–15 coupled to KLH. The synthetic analogue [Tyr¹²]-thymosin ₄ (1–15) was used as tracer. This radioimmunoassay, with a useful range of 10–1000 pmoles, showed cross-reactivity with the second homologous -thymosin of man and rat (thymosin ₁₀) but not of calf (thymosin ₉). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin , was employed for the measurement of the levels of thymosin ₄ and parathymosin in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of - and -thymosins.     

Die Wirkung einer Leberschädigung durch Thioacetamid auf die mikrosomale Aromatisierung von Testosteron bei der Ratte

Summary Rat liver microsomes, NADPH-regenerating system, and 1, 2-³H-testosterone have been incubated in vitro. The loss of tritium from the steroid, associated with aromatization of testosterone, was linear with time for 20 min and required NADPH. Pre-treatment of the rats with thioacetamide raised the liberation of tritium from 1, 2-³H-testosterone. The results suggest that liver damage by thioacetamide in rats may give rise to increased aromatization of testosterone.     

Self-labeling of human polymorphonuclear leucocyte myeloperoxidase with125iodine

In order to obtain a radioimmunoassay (RIA) technique for the measurement of human plasma myeloperoxidase (MPO), we purified the enzyme from polymorphonuclear granulocytes (neutrophils), and compared three methods of labeling it with¹²⁵Iodine: chloramine T, lactoperoxidase, and an original technique of self labeling based on the ability of the enzyme to oxidize and bind¹²⁵I in the presence of H₂O₂. The chloramine T technique produced a degraded protein, as well shown by a high non-specific binding of tracer to antibody. The lactoperoxidase technique did not succeed in labeling MPO with an adequate specific activity. In contrast, the self-labeling method gave a stable tracer with a specific activity of 23 Ci/gmg MPO (85 MBq), a satisfactory level of immunoreactivity, and a low-specific binding (3%). After labeling, purification of tracer was achieved by gel filtration chromatography in phosphate buffer (0.05 M; pH7) to which 0.1% poly-L-lysine was added. The labeled molecule remained stable for 40 days and could be used for RIA with a polyclonal antibody raised in rabbits.     

Nitrogenase activity in a transfilter culture of rhizobia with a non-leguminous plant callus culture: Transfer of fixed15N2 from bacteria toPortulaca callus

Summary A transfilter culture of the non-leguminous plantPortulaca grandiflora var. JR andRhizobium sp. cowpea 32H1 was established. Using¹⁵N₂-analysis we demonstrated that¹⁵N-containing substances produced by the bacteria passed through the membrane and¹⁵N was enriched in the plant cells.Acknowledgment. The authors wish to express their gratitude to Prof. H. Marschner and Dr G. Hentschel for the determination of the nitrogen-content of our samples by the Dumasmethod and for advice on the optic emission method. Drs J. Burton, Milwaukee, Wisc., and Tjepkema, Oregon State University, are thanked for generous gifts ofRhizobium sp. cowpea 32H1. This work was supported by the DFG.     

Thrombospondin co-localises with TGFβ and IGF-I in the extracellular matrix of human osteoblast-like cells and is modulated by 17β estradiol

Thrombospondin (TSP) is a multifunctional glycoprotein which is synthesised by several cell types including osteoblasts, and incorporated into the extracellular matrix (ECM) of these cells. The function and regulation of TSP in bone is not clear. In this study, using a long term culture model of human osteoblast-like cells, we examined the distribution of TSP in the ECM and its modulation by added estradiol. In this model the osteoblast-like cells form a regular multilayer which continues to increase in depth up to 50 days post confluence. In the ECM of these cultures and in 19-week fetal bone, the bone markers osteocalcin and alkaline phosphatase were diffusely distributed in the matrix. In contrast, labelling for TSP was concentrated, confined to the banded collagen and its immediately adjacent ECM. This pattern of labelling resembled that of the growth factors transforming growth factor-I (TGF), and insulin-like growth factor-I (IGF-I), with which TSP label co-localised. Labelling intensities were comparable between fetal bone and the in vitro material for TSP, TGF and IGF-I. TSP label was present by 10 days post confluence, reached a maximum by 20 days, and declined slowly thereafter, a time course which was similar to that of IGF-I. Incubation of osteoblast-like cell cultures with 17 estradiol resulted in an increase in multilayer depth and a maximal 3-fold increase in TSP labeling at 30 days as well as approximately 2-fold increases for TGF and IGF-I. The dose-response relationship for these responses to estradiol treatment was biphasic with maximal increases at 10^–10 M–10^–11 M of added estradiol. Treatment with 17 estradiol produced labelling intensities that were not significantly different from controls. Studies with other cell types have suggested that TSP may be involved in modulation of growth factor activity. The similarities between TSP, TGF and IGF-I, in terms of their distribution and regulation by 17 estradiol treatment, may indicate a role for TSP in modulating bone cell proliferation and function through interaction with local growth factors.     

Unterschiedliche Empfindlichkeit verschiedener Angiotensinderivate gegen Abbau durch Plasma oder Nierenhomogenat

Summary -Asp¹-Angiotensin II had a more pronounced action on the blood pressure of nephrectomized rats than the corresponding-compound.-Asp¹-Angiotensins were more slowly destroyed by rat serum, rat kidney homogenate, and human plasma than-compounds, especially-Asp¹-Angiotensin II amide.     

Metabolic pathways of δ-aminolaevulinic acid inRhodopseudomonas spheroides

Summary Incorporation of -aminolaevulinic acid 5¹⁴C and 4¹⁴C into the inosine monophosphate pool and into porphyrins, was studied in cell suspensions ofR. spheroides. The results contradict a direct incorporation of -aminolaevulinic acid into the purine ring of nucleotides through -dioxovaleric acid. It would suggest a nonspecific incorporation after degradation of -aminolaevulinic acid without a transamination as a first reaction.     

Volatiles from clover headHypera meles (Fab.) and alfalfaH. postica (Gyllenhal) weevils: search for pheromones

Summary Volatile fractions of the clover head,Hypera meles (Fab.), and alfalfa,Hypera postica (Gyllenhal), weevils contained three of four boll weevil,Anthonomus grandis (Boh.), pheromone components, (Z)-3,3-dimethylcyclohexane ^1,-ethanol and (Z)- and (E)-3,3-dimethylcyclohexane-^1,-acetaldehyde. Also found were eight oxygenated monoterpenes, previously identified as precursors and intermediates of the boll weevil pheromones.     

Identification of (3R,4R)-gD 1(6) as an isolation artefact of cannabinoid acids formed by callus cultures ofCannabis sativa L

Summary For the first time we have isolated a major psychoactive cannabinoid, (3R, 4R)- ¹⁽⁶⁾-tetrahydrocannabinol3 from callus cultures ofCannabis sativa L.3 was obtained as an artefact of the actually formed (3R, 4R)- ¹-tetrahydrocannabinol-3- and/or 5-carboxylic acids1 and2 by subjecting the culture material to a decarboxylation step prior to extraction. No attempt was made to isolated acids1 and2. The identity of3 was confirmed by comparison with an authentic sample of (3R, 4R)- ¹-tetrahydrocannabinol. Culture conditions, isolation procedure and identification of the cannabinoid are described.Acknowledgment. The authors are greatly indebted to Dr W. Heller and Dr G. Schroeder-Frey for deriving the callus cultures from seedlings ofC. sativa and to Prof. N. Amrhein for valuable gifts of culture media and growth regulators.     

Role of phospholipids in the binding activity of vasoactive intestinal peptide receptors

Summary Phospholipase digestion of rat intestinal epithelial cell membranes was performed in order to study the influence of membrane phospholipids on the binding activity of VIP receptors. Phospholipases A₂ and C strougly (ED₅₀4×10^–2 and 4×10^–1 g/ml, respectively) and rapidly reduced¹²⁵I-VIP binding to membranes whereas phospholipase D was ineffective. This suggests an important role of both hydrophobic and hydrophilic groups of phospholipids on VIP receptor binding activity.This work was supported by INSERM (CRL 827017) and the Fondation pour la Recherche Médicale Française.     

Asscorbic acid portects against male infertility in a teleost fish

An animal unable to synthesize ascorbic acid uniquely minicks human and non-human primates. Therefore, in this study we used the rainbow trout, a teleost fish, as the model animal to study the importance of dietary ascorbic acid on the fertilizing ability of sperm. A high concentration of ascorbic acid in semen plays a key role in maintaining the genetic integrity of sperm cells, by preventing oxidative damage to sperm DNA. This study will show that the concentration of asorbic acid in seminal plasma refelcts the dietary fed either an ascorbate-free diet (from 4.74±0.9 to 0.16±0.08 g ml^–1) or an ascorbate-rich diet (from 37.9±4.7 to 17.7± 3.2 g ml^–1) during the sperimnation season. The relationship between ascrobate status and fertility was studied in six groups of fish fed graded levels of ascorbic acid, which sperimated over a 150-day-period. Sperm from individual males was used to fertilize several batches of eggs. When the seminal plasma ascorbate concentration decreased to 7.3 g ml^–1 a significant decrease of fertilization rate and the hatching rate of embryos resulted. This is the first evidence that dietary ascorbate level directly affected sperm quality and influenced male fertility in a scruvy-prone vertebrate.     

Beweis des Steringerüstes von Ouabagenin

Summary Ouabagenin (I) has been degraded stepwise to the known 3-methoxy-17-carbomethoxy-^1,3,5:10-estratriene (XII). Thus the presence of a normal steroid skeleton and the -configuration of the butenolidering at C-17 is proven. The 14-configuration of the hydroxyl at C-14 was also proven. As a result of these and earlier findings, structure I can be definitely assigned to ouabagenin.

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Glykoside und Aglykone, 178. Mitteilung.     

The uptake of iodinated protein by spleen cells of guinea-pigs in vitro

Zusammenfassung 6×10^–10 g bis 10^–4 g J 125 Rinderserum-Albumin (BSA) bzw. 1,0×10^–7 bis 1,0×10^–6 g J 125 Rinderserum--Globulin (BGG) werden 3 × 10⁷ Milzzellen normaler Meerschweinchen in vitro angeboten. Unabhängig von der Höhe des Angebotes beträgt die Menge des aufgenommenen BSA 1%, die des aufgenommenen BGG 10% des Angebotes. Durch 4C Inkubations-temperatur wird die Aufnahme z.T. behindert. Die Aufnahme findet nach wenigen Sekunden Inkubationszeit statt und ist nach etwa 2–3 min abgeschlossen.

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The work was supported by a grant of the Stiftung Volkswagenwerk and of the Deutsche Forschungsgemeinschaft.     

The differentiation ofCoptis plants in vitro from callus cultures

Zusammenfassung Kalluskulturen aus Petiolastücken vonCoptis japonica (Ranunculaceae) synthetisieren das Alkaloid Berberin. Unter geeigneten Bedingungen werden auf den Kallusstücken ganze Pflanzen herangebildet.

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Part XIII in the series Studies on Plant Tissue Cultures. Part XII, see reference³.