Targeting Natural Killer Cell Reactivity by Employing Antibody to NKp46: Implications for Type 1 Diabetes

Natural killer (NK) cells belong to the innate lymphoid cells. Their cytotoxic activity is regulated by the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that the NKp46 receptor is involved in the development of type 1 diabetes (T1D). Subsequently, we hypothesized that blocking this receptor could prevent or hinder disease development. To address this goal, we developed monoclonal antibodies for murine NKp46. One mAb, named NCR1.15, recognizes the mouse homologue protein of NKp46, named Ncr1, and was able to down-regulate the surface expression of NKp46 on primary murine NK cells following antibody injection in vivo. Additionally, NCR1.15 treatments were able to down-regulate cytotoxic activity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, non-obese diabetic mice and low-dose streptozotocin. Our results show a significantly lower incidence of diabetic mice in the NCR1.15-treated group compared to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests a novel treatment strategy for early insulitis.     

Alkaloid N-oxides as transport and vacuolar storage compounds of pyrrolizidine alkaloids in Senecio vulgaris L

Cell-suspension cultures of pyrrolizidinealkaloid-producing species selectively take up and accumulate senecionine (sen) and its N-oxide (sen-Nox). Cultures established from non-alkaloid-producing species are unable to accumulate the alkaloids. The uptake and accumulation of ¹⁴C-labelled alkaloids was studied using a Senecio vulgaris cell-suspension culture as well as protoplasts and vacuoles derived from it. The alkaloid uptake exhibits all characteristics of a carrier-mediated transport. The uptake of sen-Nox follows a multiphasic saturation kinetics. The K_m-values for sen Nox of 53 M and 310 M are evaluated. Senecionine competitively inhibits sen-Nox uptake, indicating that the tertiary alkaloid and its N-oxide share the same membrane carrier. The N-oxide of sen shows a pH optimum below 5.5, whereas sen is taken up over a range from pH 4 to 8. Activation energies of 90 and 53 kJ·mol^-1 are calculated for sen-Nox and sen transport, respectively. At concentrations of 10 to 100 M, sen-Nox is rapidly taken up by cells and protoplasts; within 2 h >90% of total N-oxide is within the cells. By contrast the uptake of sen is less efficient. Vacuoles isolated from protoplasts preloaded with sen-Nox totally retained the alkaloid N-oxide, whereas sen is rapidly lost during the procedure of vacuole preparation. N-oxidation converts the weak lipophilic tertiary base into a charged polar molecule which is excellently adapted to serve as the cellular transport and storage form of pyrrolizidine alkaloids.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP 2,4-dinitrophenol - sen senecionine - sen-Nox senecionine N-oxide     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Formation of hydrogen peroxide by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.)

Summary Isolated cell-wall suspensions from horseradish in the presence of 5×10^-4 M MnCl₂ catalyze the production of hydrogen peroxide at the expense of either NADPH or NADH. This reaction is inhibited by scavengers of the superoxide free radical ion such as ascorbate or dihydroxyphenols or by superoxide dismutase, and stimulated by monophenols such as p-coumaric acid. On comparison with isolated (commercial) horseradish peroxidase it becomes evident that (a) cell-wall-bound peroxidase(s) is (are) responsible for the production of hydrogenperoxide, involving the superoxide free radical ion as an intermediate of the complex reaction chain.Abbreviation SOD superoxide dismutase     

Sexuelle Pr?gung als m?glicher Faktor innerartlicher Isolation beim Zebrafinken

Zusammenfassung An zwei Farbformen des australischen Zebrafinken(Taeniopygia guttata castanotis) sollte die Frage geklärt werden, wie sich eine in früher Jugend erfolgte Prägung auf eine bestimmte Gefiederfarbe unter Bedingungen auswirkt, die der natürlichen Lebensweise der Art weitgehend Rechnung tragen. Hierzu wurden in zwei dreiwöchigen Versuchen jeweils je 16 und 16 beider Farbformen (wildfarben und weiß), von denen jeweils 50% von der eigenen und 50% von der anderen Form aufgezogen worden waren, in einer großen Freivoliere beobachtet und ihre Paarbildung und die Orientierung ihrer sexuellen Verhaltensweisen verfolgt.Alle balzten von Anfang an unabhängig von ihrer eigenen Gefiederfarbe fast ausschließlich solche an, die der Farbe ihrer Aufzuchteltern entsprachen. Ebenso reagierten auch die nur auf das Balzverhalten von des prägungsmäßig richtigen Farbtyps. Innerhalb von drei Tagen waren 61 Vögel prägungsgemäß, und nur ein Vogel war nicht prägungsgemäß verpaart; zwei Vögel blieben unverpaart. (Die ungeraden Zahlen sind darauf zurückzuführen, daß bei einem Paar das prägungsgemäß, das jedoch nicht prägungsgemäß verpaart war). Das Verhalten der Vögel und die Sozialstruktur der Gruppe waren völlig natürlich und stimmten mit den aus Freilandbeobachtungen bekannten Einzelheiten überein. Einunddreißig Paare legten Eier und zeigten normales Brutverhalten. Zwischen den auf weiße und den auf wildfarbene Gefiederfarbe geprägten Vögeln waren keine Unterschiede (z. B. in der Strenge der Prägung) erkennbar.Die Ergebnisse lassen folgende Schlußfolgerungen zu: Prägung auf die Gefiederfarbe der Aufzuchteltern kann zu einer völligen sexuellen Isolation zwischen den Individuen mit unterschiedlicher sozialer Früherfahrung führen; eine solche Isolation wird durch das Verhalten beider Geschlechter gewährleistet; es besteht keine erfahrungsunabhängige Bevorzugung der natürlichen Gefiederfarbe der Art.

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The possible role of sexual imprinting in intraspecific sexual isolation in Zebra Finches

Summary Two color morphs (wild color and white) of the Australian Zebra Finch (Taeniopygia guttata castanotis) were used to examine the influence of early imprinting on plumage color with respect to mate selection during pair formation, under semi-natural conditions. Two experiments, each of three weeks duration, were conducted in a large outdoor flight cage. A total of 32 birds (16 and 16 ) of both color morphs were used in each experiment. Half of the birds in each color group were raised by their own parents and the other half were hatched and raised by foster parents of the other color morph. Orientation of all courtship behavior as well as actual pair formation was observed.From the onset the directed their courtship behavior almost exclusively towards of their (foster) parents color morph and the same positive orientation towards plumage coloration was observed in the . Within three days 61 of the 64 birds, irrespective of their own plumage color, had chosen mates which were consistent with their early experience, while only one bird was paired with the wrong color morph and two birds remained unpaired. (The uneven numbers are a result of the fact that in one pair the was paired consistent with its early experience while the was not). All aspects of the sexual behavior of the birds and the social structure of the group were in agreement with results obtained during previous field observations in Australia. During the course of the experiment thirty-one pairs produced clutches and showed normal incubation behavior. No difference in the strength of the imprinting response was found between birds raised by wild color or white parents.The conclusions drawn from these results were that imprinting on the plumage color of the (foster) parent may lead to complete sexual isolation between individuals of different early experience. Such isolation is achieved and maintained by the behavior of both sexes. Finally, there seems to be no innate preference for the natural plumage color of the species (wild color).

     

Genetic analysis of the X-chromosomal region 1E-2A of Drosophila melanogaster

Reversion mutagenesis of three single P elements located in the cytogenetic interval 1E-2A at the tip of the X chromosome of Drosophila melanogaster was used to recover new deletions in this chromosomal region. The deletions obtained include small aberrations within region 2A and larger lesions extending from 2A into 1E and 1B. All three screens also yielded terminal deficiencies. The new deficiencies, together with previously characterized rearrangements, were analyzed for their complementation behaviour with the maternal effect locus fs(1)Nasrat and lethal loci in the region. These analyses provide an overall genetic map of the interval 1E-2A. In addition, the smaller deletions were physically mapped within cloned genomic DNA of the 2A region.     

The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo

Pemphigus foliaceus (PF) is a life-threatening autoimmune blistering skin disease caused by pathogenic IgG autoantibodies against desmoglein 1 (dg1), a desmosomal cadherin-type adhesion glycoprotein. Using lectins and glycosidases, we have shown that dg1 displays an N-glycosylation pattern of the complex triantennary type. We have found that lectins and glycosidases interfere with N-bound sugar residues on the amino-terminal ectodomain of dg1 and completely abolish, in vitro, the antigenicity of dg1 in most of the patients' sera. Moreover, in an ex vivo model using punch biopsies from normal human skin, we demonstrate that preincubation of the epidermis in wheat germ agglutinin (WGA) prevents PF autoantibody binding, acantholysis, and subcorneal blistering. In addition, we show that topical treatment with WGA inhibits PF autoantibody binding to keratinocytes in both newborn BALB/c mice and in organotypic human epidermis grafted onto the back of SCID mice. The epidermis of these pretreated animals displays a regular morphology, whereas control animals develop the immunopathologic phenotype of PF. These findings suggest that WGA may interfere with autoantibody binding to dg1, preventing experimental PF without affecting the adhesive function of dg1. Our observations may provide a new approach to the therapy of PF.     

A gene mutated in nephronophthisis and retinitis pigmentosa encodes a novel protein,nephroretinin, conserved in evolution

Nephronophthisis (NPHP) comprises a group of autosomal recessive cystic kidney diseases, which constitute the most frequent genetic cause for end-stage renal failure in children and young adults. The most prominent histologic feature of NPHP consists of development of renal fibrosis, which, in chronic renal failure of any origin, represents the pathogenic event correlated most strongly to loss of renal function. Four gene loci for NPHP have been mapped to chromosomes 2q13 (NPHP1), 9q22 (NPHP2), 3q22 (NPHP3), and 1p36 (NPHP4). At all four loci, linkage has also been demonstrated in families with the association of NPHP and retinitis pigmentosa, known as “Senior-Løken syndrome” (SLS). Identification of the gene for NPHP type 1 had revealed nephrocystin as a novel docking protein, providing new insights into mechanisms of cell-cell and cell-matrix signaling. We here report identification of the gene (NPHP4) causing NPHP type 4, by use of high-resolution haplotype analysis and by demonstration of nine likely loss-of-function mutations in six affected families. NPHP4 encodes a novel protein, nephroretinin, that is conserved in evolution—for example, in the nematode Caenorhabditis elegans. In addition, we demonstrate two loss-of-function mutations of NPHP4 in patients from two families with SLS. Thus, we have identified a novel gene with critical roles in renal tissue architecture and ophthalmic function.     

Very long-chain phenylpropyl and phenylbutyl esters from Taxus baccata needle cuticular waxes

The cuticular wax of Taxus baccata L. needles was found to contain four different classes of long-chain esters that were identified by various chemical transformations with product assignment employing GC-MS. Homologous series of (1) 3-(4'-hydroxyphenyl)-propyl esters of C(20)-C(36) fatty acids, (2) 4-(4'-hydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids, (3) 3-(3',4'-dihydroxyphenyl)-propyl esters of C(20)-C(32) fatty acids, and (4) 4-(3',4'-dihydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids were identified. The four compound classes amounted to 0.1-3.6 micro g/cm(2) of needle surface area, corresponding to 0.2-7.6% of the wax mixture, respectively. While both phenylpropyl ester series had a maximum for the homolog containing tetracosanoic acid, in the phenylbutyl esters homologs containing eicosanoic and docosanoic acids predominated.