Anti-inflammatory action of IL-4. Negative regulation of contact sensitivity to trinitrochlorobenzene.

We examined the effects of IL-4, a cytokine produced by Th2 cells, on the development of an Ag-specific, T cell-mediated inflammatory response in a hapten-induced model of contact sensitivity (CS). Intravenous administration of IL-4 was ineffective in modulating the development of CS when administered on days 0, 1, and 2 after sensitization with the hapten trinitrochlorobenzene. In contrast, such treatment significantly reduced the response when given on the day of challenge. Conversely, treatment with anti-IL-4 mAb on day 4 markedly increased the magnitude of CS but was without effect when administered on days 0, 1, and 2. These results suggest that IL-4 interferes with CS at the efferent but not the afferent limb of the response. IL-4 had no inhibitory effect on the ability of immune lymph node cells to transfer adoptively CS or their proliferation upon restimulation with hapten. However, the expression of CS by immune cells was severely curtailed in mice treated with IL-4 prior to immune cell transfer. Furthermore, IL-4 inhibited monokine (gamma-IFN inducible protein [IP-10] and TNF-alpha) expression in macrophages induced by treatment with culture supernatants from the Ag-stimulated immune lymph node cells. These results indicate that suppression of Ag-specific inflammatory CS response by IL-4 may be mediated at least in part through inhibition of cytokine production by mononuclear phagocytes infiltrating the site.     

Immunohistochemical expression of rabphilin-3A-like (Noc2) in normal and tumor tissues of human endocrine pancreas

Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans.     

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular basis of recognition by Gal/GalNAc specific legume lectins: influence of Glu 129 on the specificity of peanut agglutinin (PNA) towards C2-substituents of galactose

The ability to discriminate between galactose and N- acetylgalactosamine, observed in some lectins, is crucial for their biological activity as well as their usefulness as tools in biology and medicine. However, the molecular basis of differential binding of lectins to these two sugars is poorly understood. Peanut agglutinin (PNA) is one of the few galactose-specific legume lectins which does not bind N- acetylgalactosamine at all and is, therefore, ideal for the study of the basis of specificity towards C-2 substituted derivatives of galactopyranosides. Examination of the three-dimensional structure of PNA in complex with lactose revealed the presence of both a longer loop and bulkier residues in the region surrounding the C-2 hydroxyl of the galactopyranoside ring, which can sterically prevent the accommodation of a bulky substituent in this position. One such residue, is a glutamic acid at position 129 which protrudes into the binding site and perhaps directly obstructs any substitution at the C-2 position. Two mutants in bacterially expressed PNA were therefore constructed. These were E129D and E129A, in which Glu129 was replaced by Asp and Ala, respectively. The specificity of the mutants for galactose, galactosamine, and N- acetylgalactosamine was examined through observing the inhibition of hemagglutination and binding of the lectin to immobilized asialofetuin. The results showed that the affinity of E129A and E129D for C-2-substituted derivatives of the galactose varies. The mutant E129D showed significant binding towards N- acetylgalactosamine, suggesting that the residue Glu 129 is crucial in imparting exclusive galactose-specificity upon PNA. This study not only attempts to provide an explanation for the inability of PNA to accommodate C-2-substituted derivatives at its primary subsite, but also seeks to present a basis for engineering lectins with altered specificities.      

Reconstructing Indian-Australian phylogenetic link

Background  

An early dispersal of biologically and behaviorally modern humans from their African origins to Australia, by at least 45 thousand years via southern Asia has been suggested by studies based on morphology, archaeology and genetics. However, mtDNA lineages sampled so far from south Asia, eastern Asia and Australasia show non-overlapping distributions of haplogroups within pan Eurasian M and N macrohaplogroups. Likewise, support from the archaeology is still ambiguous.     

Differences in murine gut-associated lymphoid tissues in generating broadly nonspecific cytotoxic cells in response to interferon alpha A/D and interleukin 2

We examined the response of cells of murine gut-associated lymphoid tissues to agents that augment the activity of natural killer (NK) cells. Specifically, we studied the effect of polyinosinic: polycytidylic acid (Poly I:C) in vivo, and recombinant interferon alpha A/D (rIFN alpha A/D) and recombinant interleukin 2 (rIL2) in vitro on lymphoid cells of mesenteric lymph nodes (MLN) and Peyer's patches (PP) in generating cytotoxicity against NK-sensitive (YAC-1) and NK-insensitive (B16BL6) tumor targets. The effect of these agents on spleen cells was examined for comparison with their effect on MLN and PP cells and as a positive control. MLN and PP cells lacked spontaneous NK activity: however, NK activity could be augmented to different levels by the three agents. The treatment of mice in vivo with Poly I:C induced considerable cytotoxicity in the spleen and MLN but only a weak cytotoxic response in PP. The in vitro enhancement of NK activity by rIFN alpha A/D was strong in the spleen, intermediate in MLN, and consistently poor in PP. The weak NK augmentation by rIFN alpha A/D in PP was not restricted to a single mouse strain. PP cells from five strains of mice responded poorly to rIFN alpha A/D. Furthermore, NK augmentation by rIFN alpha A/D in PP cells did not improve after passing the responder cells through nylon wool, indicating that the lack of augmentation of NK activity was not the result of a preponderance of B cells or the masking of NK cells by adherent lymphoid populations in PP. In contrast to weak augmentation of NK activity by rIFN alpha A/D, considerable IL2-induced lymphocyte-activated killer (LAK) activity against NK-insensitive B16BL6 tumor cells was induced in PP. Limiting-dilution analysis showed that the frequency of LAK precursors in the MLN and PP was not markedly different from that of the spleen. The differences among spleen, MLN, and PP lymphoid populations in generating the broadly nonspecific cytotoxic effector cells in response to rIFN alpha A/D or rIL2 may result from differences in the pools of different pre-NK cells or to differential sensitivity of the same pool of pre-NK cells to rIFN alpha A/D and rIL2 in different anatomical locations.     

Oral administration of the contact sensitizer trinitrochlorobenzene: initial sensitization and subsequent appearance of a suppressor population

Our earlier studies have demonstrated that intragastric administration of the hapten trinitrochlorobenzene (TNCB) 2 to 3 weeks prior to attempting sensitization with epidermally applied hapten can abrogate development of systemic contact sensitivity (CS). In this paper, we have examined whether onset of tolerance following intragastric administration of the hapten is preceded by development of hapten-specific CS. Indeed, CS was found to be present 5 days after feeding TNCB and in most experiments the response decreased significantly by Days 10 to 12. The kinetics of development of CS by the oral and epidermal routes were strikingly similar except that the magnitude of reactivity (up to 5 days) in orally sensitized mice was somewhat less than that of epidermally sensitized mice. With the exception of Peyer's patches (PP), effector cells of CS were recovered from such gut-associated lymphoid tissues as mesenteric lymph nodes (MLN), lamina propria, and lymphocytes that are present in the intraepithelial compartment of the intestinal wall. These cells as well as spleen cells of TNCB-fed mice were able to adoptively transfer CS to naive mice. The capacity of MLN and spleen cells of TNCB-fed mice to confer CS adoptively was abrogated after treating cells with anti-Thy 1.2 and anti-Lyt 1.1 antibodies plus complement thereby identifying them as T lymphocytes. Although CS decreased by 10-12 days after feeding TNCB, the decline was reversed by pretreating mice with cyclophosphamide (CY) 2 days before giving the hapten. Whereas spleen cells from animals fed hapten 5 days earlier transferred CS readily, those from mice fed hapten 12 days earlier did not. However, when 12-day spleen cells were depleted of Lyt 2+ cells their ability to adoptively transfer CS was restored. These observations indicate that feeding TNCB to mice initially produces CS, mediated by Thy 1.2+, Lyt 1.1+ lymphocytes. CS is subsequently down-regulated by activation of Lyt 2+ suppressor cells, precursors of which are sensitive to CY.     

Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts

FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast size and number.