Dimerization of NKp46 receptor is essential for NKp46-mediated lysis: characterization of the dimerization site by epitope mapping

NKp46 is a primary activating receptor of NK cells that is involved in lysis of target cells by NK cells. Previous studies showed that the membrane-proximal domain of NKp46 (NKp46D2) retained the binding of NKp46 to its ligands and is involved in lysis. We studied NKp46D2 by using a peptide-based epitope mapping approach and identified an NKp46D2-derived linear epitope that inhibited NKp46-mediated lysis. The epitope, designated as pep4 (aa 136-155), interacted with NKp46, and lysis by NK cells was inhibited by the presence of pep4. Through modeling and mutagenesis, we showed that pep4 could be involved in NKp46 homodimerization. R145 and D147 contribute to the function of pep4, and R145Q mutation in recombinant NKp46 reduced its binding to target cells. At the cellular level, fluorescent resonance energy transfer analysis revealed that pep4 is indeed involved in dimerization of cell membrane-associated NKp46. We suggest that the NKp46-derived pep4 site is part of the dimerization surface of NKp46 and that NKp46 dimerization contributes to NKp46-mediated lysis by NK cells.     

The trans-anethole degradation pathway in an Arthrobacter sp.

A bacterial strain (TA13) capable of utilizing t-anethole as the sole carbon source was isolated from soil. The strain was identified as Arthrobacter aurescens based on its 16 S rRNA gene sequence. Key steps of the degradation pathway of t-anethole were identified by the use of t-anethole-blocked mutants and specific inducible enzymatic activities. In addition to t-anethole, strain TA13 is capable of utilizing anisic acid, anisaldehyde, and anisic alcohol as the sole carbon source. t-Anethole-blocked mutants were obtained following mutagenesis and penicillin enrichment. Some of these blocked mutants, accumulated in the presence of t-anethole quantitative amounts of t-anethole-diol, anisic acid, and 4,6-dicarboxy-2-pyrone and traces of anisic alcohol and anisaldehyde. Enzymatic activities induced by t-anethole included: 4-methoxybenzoate O-demethylase, p-hydroxybenzoate 3-hydroxylase, and protocatechuate-4,5-dioxygenase. These findings indicate that t-anethole is metabolized to protocatechuic acid through t-anethole-diol, anisaldehyde, anisic acid, and p-hydroxybenzoic acid. The protocatechuic acid is then cleaved by protocatechuate-4,5-dioxygenase to yield 2-hydroxy-4-carboxy muconate-semialdehyde. Results from inducible uptake ability and enzymatic assays indicate that at least three regulatory units are involved in the t-anethole degradation pathway. These findings provide new routes for environmental friendly production processes of valuable aromatic chemicals via bioconversion of phenylpropenoids.     

Desensitizing and non-desensitizing subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in cockroach neurons

Two alpha-bungarotoxin-sensitive nicotinic receptor subtypes in cockroach neurons are identified as desensitizing (nAChD), selectively inhibitable with 100 nM imidacloprid, and non-desensitizing (nAChN), selectively inhibitable with 100 pM methyllycaconitine. Although the desensitization rate of nAChD receptors is highly variable, pharmacology is largely independent of desensitization rate. Because desensitized states tightly bind agonists, nAChD receptors are potently inhibited by neonicotinoids and specifically measured in radiolabeled imidacloprid binding assays. However, they are not usually detected in binding assays with radiolabeled alpha-bungarotoxin, which has a Kd for the resting state of 21 nM, but binds poorly to desensitized states often present in binding assays. In contrast, nAChN receptors are specifically measured in binding assays with radiolabeled alpha-bungarotoxin, which binds them with a Kd of 1.3 nM. nAChN receptors are activated by neonicotinoids at micromolar concentrations, and allosterically by spinosyn A, with an EC50 of 27 nM. Spinosyn A weakly antagonizes nAChD receptors -23% at 10 microM. The roles of the two nAChR subtypes in insecticide poisoning are discussed.     

Development and application of a DNA microarray-based yeast two-hybrid system

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms.     

Breeding systems in thePolemoniaceae

Pollen — ovule ratios and percentages of stigmatic pollen germination (SPG) were measured for over 160 taxa of thePolemoniaceae. When related to taxa with known breeding systems, it is found that low SPGs and high P:O ratios characterize xenogamous plants, high SPGs and low P:Os characterize autogamous plants. There is a significant negative correlation between P:O ratio and SPG in the whole family as well as in certain genera. Both measures can serve as reliable indicators of the breeding systems in taxa of thePolemoniaceae and can be measured in dried or living specimens. Accordingly, most polemons feature mixed breeding system, i.e. facultative xenogamy or facultative autogamy. Xenogamy is common among the tropical genera and in theLeptodactylon, Phlox andPolemonium. Autogamy is more frequent in the tribeGilieae (particularly inGilia) than in the other tribes. Annual taxa tend to be autogamous, showing on the average higher SPG and lower P:O ratio. The lepidopteran-pollinated group of species have a higher mean P:O ratio and lower mean SPG thus indicating that such plants are associated with crosspollination more than the others.     

Spontaneous DNA repair in human peripheral blood mononuclear cells

DNA molecules are constantly damaged during mitosis and by oxygen-free radicals produced by either cellular metabolism or by external factors. Populations at risk include patients with cancer-prone disease, patients under enhanced oxidative stress, and those treated with immunosuppressive/cytotoxic therapy. The DNA repair process is crucial in maintaining the genomal DNA integrity. The aim of this study was to evaluate spontaneous DNA repair capacity of peripheral blood mononuclear cells (PBMC) from normal blood donors. PBMC DNA repair ability represents DNA repair by other tissues as well. It is shown in the present study that in vitro incorporation of [3H]thymidine in non-stimulated PBMC expresses the ability of the cells to repair DNA damage. This method was validated by double-stranded DNA measurements. Both catalase and Fe2+ increased DNA repair, the former by preventing re-breakage of newly repaired DNA and the latter by introducing additional DNA damage, which enhanced DNA repair. Better understanding of DNA repair processes will enable to minimize DNA damage induced by oxidative stress.     

Locating alignments with k differences for nucleotide and amino acid sequences

Given two sequences, a pattern of length m, a text of lengthn and a positive integer k, we give two algorithms. The firstfinds all occurrences of the pattern in the text as long asthese do not differ from each other by more than k differences.It runs in O(nk) time. The second algorithm finds all subsequencealignments between the pattern and the test with at most k differences.This algorithm runs in O(nmk) time, is very simple and easyto program. Received on August 12, 1987; accepted on December 31, 1987     

Translocations between Eu- and heterochromatic chromosomes,and spermatocytes lacking a heterochromatic set in male mealy bugs

In male mealy bugs the chromosomes of paternal origin become heterochromatic (H) early in embryogeny while those of maternal origin remain euchromatic (E). First instarPseudococcus obscurus Essig males which were irradiated with 3000 r of X-rays carried translocations between E and H chromosomes [T(E;H)'s] in their spermatocytes. In the T(E;H)'s the border between the E and H segments was usually quite sharp, but occasionally short E segments may have become either partially or completely heterochromatic. During the second meiotic division the E and H sets normally segregate to opposite poles. The T(E;H)'s often formed bridges in AII and TII, but in most of the cells they did succeed in reaching one of the poles. The segregation of the T(E;H)'s depended on the relative size of their E and H segments. When the E and H segments were of the same size, the T(E;H)'s segregated more often with the E chromosomes, even though the latter have been observed to be attached to their pole with fewer spindle fibers. Thirty-five of the 173 males analyzed had sectors in their testes which lacked an H set. The number of cysts per sector suggested that each sector was derived from a single irradiated cell. The karyotypes observed in some of the sectors indicated that the lack of an H set was the result of reversal of heterochromatization and not due to the loss of the H set and the endoduplication of the E set. Most of the cells lacking an H set divided normally during the first meiotic division. The second division, however, was abortive and resulted in the production of diploid sperm. Two possibilities for the origin of spermatocytes lacking an H set are considered: (i) that these spermatocytes resulted from X-ray induced reversal of heterochromatization in spermatogonia, and (ii) that these spermatocytes originated from presumptive cyst wall cells whose H set had undergone reversal prior to the irradiation.Supported by grant GB 6745 from the National Science Foundation, Washington, D. C.     

Undercondensation and localized euchromatinization of the X chromosome in the grasshopper Melanoplus femur-rubrum

In most animals, including grasshoppers, the X chromosome is heterochromatic (heteropycnotic) during prophase I and metaphase I of spermatogenesis. This report describes one grasshopper male in which at these states some of the X chromosomes contained an euchromatic (E) segment. In grasshoppers, the heteropycnotic state of the X is apparently established prior to the formation of the cysts. The spermatocytes containing the E segments, however, did not comprise whole cysts. It was concluded, therefore, that the E segments resulted from a localized euchromatinization rather than a failure to become heteropycnotic. The cytology of this male was unusual in two other respects. In most of the spermatocytes the chromosomes were longer and thinner than those of other males. In addition, in some of the cells undergoing meiosis, the cytoplasm failed to divide during both meiotic divisions and the resulting spermatids failed to differentiate into sperm. Because in this species both the presence of Xs with E segments and undercondensation are very rare and both involve condensation, it is likely that they are in some way related. Evidence for and against the possibility that the E segments were genetically active and that this activity led to the arrest of some of the spermatids is discussed.     

Nonreplication of heterochromatic chromosomes in a mealy bug,Planococcus citri (Coccoidea: Homoptera)

In males of mealy bugs with the lecanoid chromosome system, the paternal set of chromosomes becomes heterochromatic in early embryogeny. In males of the mealy bug, Planococcus citri, the heterochromatic (H) set in testis sheath cells and in most of the oenocytes apparently did not replicate while the euchromatic (E) set was undergoing several cycles of endoreplication. In third instar males, testis sheath cells in endoanaphase and endotelophase exhibited 5H and either 40 or 80E chromosomes. The increase in the number of E chromosomes was attributed to the replication of only the E chromosomes. Oenocytes of third instar males had 0, 5, or 10H chromosomes and from 10 to 240E chromosomes. The oenocytes with 5H chromosomes had a mean of 50.8E chromosomes, and those with 10H chromosomes had a mean of 155.6E chromosomes. Nuclear and cell fusion was considered as a means of producing the various numbers of H and E chromosomes in oenocytes, and it was concluded that although nuclear fusion probably took place, the differences between the number of H and E chromosomes was at least in part due to replication of only the E chromosomes. The size of the H chromosomes was about the same in all the testis sheath cells and the oenocytes irrespective of the level of endopolyploidy for the E set. These H chromosomes apparently did not increase in polyteny, because they were only about half the size of the H chromosomes in prophase I of spermatogenesis. The significance of the nonreplication of the H set and the control of nonreplication are briefly discussed.This study was aided by a grant (GB-1585) from the National Science Foundation, Washington, D.C.