Silver Nanoparticle Films Obtained by Convective Self-Assembly for Surface-Enhanced Raman Spectroscopy Analyses of the Pesticides Thiabendazole and Endosulfan.

Pesticides pose a great threat to human health and their rapid detection has become an urgent public safety issue engaging the scientific community to search for fast and reliable detection techniques. In this context, Surface Enhanced Raman Spectroscopy (SERS) has emerged as a valuable detection and analysis tool due to its high sensitivity and selectivity, proving its suitability for the food industry and environmental monitoring applications. Here, we report on the fabrication of colloidal silver nanoparticle (AgNP) films by convective self-assembly (CSA) on solid planar substrate and their use for the SERS analyses of two types of pesticides, the fungicide thiabendazole (TBZ) and the insecticide α-endosulfan (α-ES). Electron microscopy shows that these nanoparticle films are dense, highly compact, and uniform across several mm2 areas. The SERS efficiency of the fabricated AgNP films is evaluated using a well-known Raman probe, p-aminothiophenol, for multiple excitation laser lines (532 nm, 633 nm, and 785 nm). The films exhibit the largest SERS enhancement factors for 785 nm excitation, reaching values larger than 105. Thiabendazole could be readily adsorbed on the AgNPs without any sample surface functionalization and detected down to 10-6 M, reaching the sub-ppm range. Endosulfan, a challenging analyte with poor affinity to metal surfaces, was captured near the metal surface by using self-assembled alkane thiol monolayers (hexanethiol and octanethiol), as demonstrated by the thorough vibrational band analysis, and supported by density functional theory (DFT) calculations. In addition, principal component analysis (PCA) based on SERS spectra offers significant leverage in discrimination of the molecules anchored onto the metallic nanostructured surface. This present study demonstrates the utility of self-assembled colloidal nanoparticle films as SERS substrates for a broad range of analytes (para-aminothiophenol, thiabendazole, α-endosulfan, and alkanethiols) and contributes to the development of SERS-based sensors for pesticides detection, identification and monitoring.

Defective development of NK1.1+ T-cell antigen receptor alphabeta+ cells in zeta-associated protein 70 null mice with an accumulation of NK1.1+ CD3- NK-like cells in the thymus.

Development of natural killer 1.1+ (NK1.1+) CD3+ (NK1.1+ T) cells was analyzed in zeta-associated protein 70 (ZAP-70) null ((-/-)) mice. Both NK1.1+ TCRalphabeta+ and NK1.1+ TCRgammadelta+ cell populations were absent in the thymus and spleen. By contrast, the number of NK1.1+ CD3- cells was increased in these tissues. The NK1.1+ CD3- thymocytes in ZAP-70(-/-) mice had surface phenotypes in common with NK or NK1.1+ T cells. However, some of them were discordant either with NK cells or with NK1.1+ T cells. The NK1.1+ CD3- cells produced interferon-gamma upon stimulation with NK1.1 cross-linking in the presence of interleukin-2 and exhibited a substantial cytotoxicity against YAC-1 cells. Moreover, the generation of NK1.1+ T cells with invariant Valpha14Jalpha281 chains was induced from the NK1.1+ CD3- thymocytes following stimulation with phorbol myristate acetate and ionomycin in a neonatal thymic organ culture. An introduction of TCRalpha and beta transgenes to the ZAP-70(-/-) mice resulted in generation of an NK1.1+ TCRalphabeta(dim) population, whereas no substantial CD4+ CD8- or CD4- CD8+ population that expressed the introduced TCRalphabeta was generated in the mainstream T lineage. These findings demonstrate that ZAP-70 kinase is indispensable for the development of NK1.1+ T cells and that the unique NK1.1+ CD3- thymocytes in ZAP-70(-/-) mice contain immediate precursors of NK1.1+ T cells.

Involvement of CD28/CTLA4-B7 costimulatory pathway in the development of lymphadenopathy and splenomegaly in MRL/lpr mice.

MRL/lpr mouse is an established animal model which develops autoimmune diseases including glomerulonephritis, sialoadenitis, hepatitis and inflammatory lung disease. Additionally, it has been reported that lpr strains uniquely accumulate CD3+ CD4- CD8- B220+ (double negative, DN) T cells in lymphoid organs leading to lymphadenopathy and splenomegaly. To investigate the role of CD28/CTLA4-B7 pathway in the development of lymphadenopathy and splenomegaly, MRL/lpr mice were treated with soluble form of CTLA4 molecules, CTLA4IgG, which efficiently blocks this pathway. It was demonstrated that (i) the development of DN T cells was independent of the CD28/CTLA4-B7 pathway, (ii) the CD28/CTLA4-B7 pathway was required for the development of lymphadenopathy and splenomegaly, (iii) the CD28/CTLA4-B7 pathway was important for the accumulation of various cell populations in the lymph node and spleen, (iv) composition of the accumulating cell populations was not altered by CTLA4IgG treatment, and (v) activation of conventional T cells and IL-4 production from conventional T cells were the CD28/CTLA4-B7 pathway dependent. Thus, we concluded that the CD28/CTLA4-B7 pathway was required for the development of full-blown lymphadenopathy and splenomegaly in MRL/lpr mice.

Detection of various epitopes of murine osteopontin by monoclonal antibodies.

We immunized rats with recombinant murine osteopontin protein and obtained four monoclonal antibodies recognizing distinct epitopes of murine osteopontin. OPN1.2 recognized the amino-terminal half of OPN, while OPN2.2, OPN2.3, and OPN3.1 recognized the carboxy-terminal half of OPN. The epitope recognized by OPN2.2 was destroyed by further cleavage of the carboxy half of OPN. The epitope recognized by OPN2.3 was located in the amino-terminal end of the carboxy half of OPN, whereas that recognized by OPN3.1 was located in the carboxy-terminal end of the carboxy half of OPN. OPN1.2 and OPN2.2 recognized thrombin-cleaved osteopontin, whereas thrombin-cleaved osteopontin was not recognized by OPN2.3 and OPN3.1. Thus, these monoclonal antibodies will be useful in structure/function studies of the role of osteopontin in murine models of disease.

Haptoglobin in Carnivora: a unique molecular structure in bear, cat and dog haptoglobins.

Haptoglobin (Hp), a hemoglobin-binding protein in plasma, consists of alpha and beta subunits and has a tetra-chain arrangement (beta-alpha-alpha-beta) connected by disulfide bridges in most mammals so far examined. Dog Hp has been reported to be unique compared with other Hps in respect that (1) the two alpha beta units are joined by a non-covalent interaction rather than a disulfide bridge and (2) the alpha chain has an oligosaccharide-binding sequence (Asn-X-Ser/Thr) and is glycosylated. To determine whether the unique structures of dog Hp are common in the Carnivora, we purified Hps from sera of bear and cat, and analyzed their subunit structure and partial amino acid sequences. The analyses by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and non-reducing conditions, revealed that bear and cat Hps have similar subunit arrangements to dog Hp, suggesting the absence of a disulfide bridge between two alpha chains. This was confirmed by amino acid sequence analysis of the alpha chains: that is, Cys15 participating in the inter-alpha chain disulfide bridge was replaced by Val in bear or Leu in cat and dog. Thus, the unique subunit arrangement of Hp reported in dog may be common in the Carnivora. In contrast to dog Hp, however, alpha chains of bear and cat Hps were found not to have the typical oligosaccharide binding sequence on their alpha chains and were not glycosylated.

Stimulation of haptoglobin synthesis by interleukin-6 and tumor necrosis factor, but not by interleukin-1, in bovine primary cultured hepatocytes.

The hepatic synthesis of acute phase proteins in ruminants has been suggested to be regulated by some mechanisms different from those in other species such as rodents and human. To explore possible regulatory factors unique to ruminants, we examined effects of interleukin (IL)-6, IL-1 and tumor necrosis factor (TNF), on haptoglobin (Hp) synthesis using a primary culture system of bovine hepatocytes. After bovine primary cultured hepatocytes were incubated in the presence of various concentrations of the cytokines, the synthesis and mRNA level of haptoglobin and albumin were measured by labeling with [35S]-methionine and immunoprecipitation, and by Northern blot analysis, respectively. Hp synthesis was dose-dependently increased by recombinant human (rh) IL-6, and also by rhTNF-alpha, but to a less extent, while it was not affected by rhIL-1 beta. The stimulatory effect is mainly pretranslational, because mRNA level of Hp changed in parallel with protein synthesis. In contrast, albumin synthesis was suppressed by these three cytokines similarly. These results are inconsistent with the previously proposed view that TNF and IL-1 overlap in their pathways leading to the transcriptional activation of many acute phase protein genes. In conclusion, there is a species-specific unique signaling system, especially for TNF, in transcriptional activation of bovine Hp gene.

Isolation and primary culture of bovine hepatocytes: albumin synthesis and adrenergic activation of glycogenolysis.

We describe a technique for isolation and primary culture of bovine hepatocytes, and their metabolic characterization. Hepatocytes were isolated from the caudate lobe of bovine liver by perfusion with 0.25 mM ethylene-glycol tetraacetic acid and 0.05% collagenase. The viability and yield of the cells were 70-92% and 0.1-3.6 x 10(7) cells/g liver, respectively. When the isolated hepatocytes were cultured in Williams' medium E, they began to spread in 3 hr and formed monolayers in 24 hr. These monolayers were retained for at least 6 days. To monitor the metabolic activities specific to liver, synthesis and secretion of albumin were measured by labeling with [35S]-methionine and immunoprecipitation. This activity was low in isolated hepatocytes, but increased after culturing 1-3 days, and decreased again after 6 days. Glycogenolytic activity was also assessed by measuring glucose release to the medium by stimulation with epinephrine. The glycogenolytic response to epinephrine was also enhanced by culturing the hepatocytes 1-3 days, but was decreased after 6 days. Since the isolated bovine hepatocytes retained the liver-specific activities of albumin synthesis and glycogenolysis for several days in culture, these cells are useful for cellular and molecular studies on the functions of bovine liver.

Purification and identification of a serum protein increased by anthelmintic drugs for Dirofilaria immitis in dogs.

Polyacrylamide gel electrophoretic analysis of canine serum protein has revealed that the administration of anthelmintics elicits an increase in a certain serum protein. This protein, named PT60, was partially purified by ammonium sulfate fractionation and preparative electrophoresis. The purified PT60 gave a single band with the molecular size of 53 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. After reduction with 2-mercaptoethanol, two bands appeared at 35 kDa and 17 kDa, indicating that PT60 consists of two subunits which are linked with each other by disulfide bonds. PT60 had the capacity to bind to hemoglobin. In an immunodiffusion test, an antiserum against PT60 cross-reacted with canine haptoglobin (Hp). N-terminal amino acid sequences of two PT60 subunits were identical to those of alpha and beta subunits of canine Hp, respectively. Thus, PT60 was identified as Hp.