New Thioureas and Related Substances Intended for Melanoma Targeting

Thiouracil and a few related drugs are known to be melanoma-seeking agents owing to specific incorporation into nascent melanin. The melanin-affinic properties are apparently due to binding to intermediates, preferably dopaquinone, produced in the melanin synthetic pathway by tyrosinase-catalysed oxidation of tyrosine. In the present paper, in vitro screening methods have been used for the identification of possible melanoma seekers according to the above principle. The binding of test substance to dopaquinone suppresses dopachrome formation by the withdrawal of dopaquinone from the reaction mixture, and the decrease in dopachrome concentration was monitored spectrophotometrically at 475 nm. In order to eliminate false results caused by tyrosinase inhibition, which also will decrease the dopachrome concentration, the oxygen consumption was followed potentiometrically. To avoid the effect of tyrosinase inhibition on dopachrome formation, additional experiments with autoxidation of L-dopa in the presence of test substance were performed. Of the 22 substances (mainly thioureylenes and thioamides) studied, 4,5,6-triamino-2(H)-pyrimidinehtionsulfate, trithiocyanuric acid, 2-thiouracil, 6-methyl-2-thiouracil, and 4-amino-2-mercaptopyrimidine most effectively decreased the dopachrome formation with no or little inhibition of tyrosinase activity. They should therefore be regarded as potential melanoma seekers. In a complementary autoradiographic study on the uptake of the potent tyrosinase inhibitor mercaptobenzothiazole (MBT) in B 16 melanoma, transplanted to mice, it was found that strong tyrosinase inhibition seems to decrease incorporation into melanin in vivo. MBT was partly accumulated in restricted areas of the tumor, which may be explained by the small molar dose injected.     

Trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) mitochondrial DNA polymorphism in the centre of the marble trout distribution area

The marble trout, a lineage of the Salmo trutta complex, is endemic to the Southern Alpine region. Although it is endangered throughout its entire distribution range, population genetic data were lacking for the central area, including the upper Etsch/Adige River system (South Tyrol, Northern Italy). A total of 672 Salmo trutta specimens, comprising phenotypic marble trout and phenotypic brown trout, from 20 sampling sites throughout South Tyrol were analysed by sequencing the complete mitochondrial DNA control region. Thirteen distinct haplotypes were identified, which clustered within three major genetic lineages: the Marmoratus (MA), the Atlantic (AT) and the Danubian (DA) lineage. 41.7% of the investigated individuals carried haplotypes of the MA lineage, 47.9% of the AT lineage and 10.4% of the DA lineage. It is noticeable that AT haplotypes were present at all sampling sites and no “pure” marble trout population with exclusively MA haplotypes was found. This points to a considerable impact of stocking with allochthonous brown trout, given that there is no evidence for natural colonisation by individuals of the AT lineage. However, our data indicate, for at least four localities, a limited gene flow between the native marble trout and hatchery-reared strains. Future conservation and rehabilitation measures will thus have to concentrate on the identification of remnant pure marble trout individuals from such mixed populations. Handling editor: C. Sturmbauer     

Botryoid odontogenic cyst. Exploration of proliferative activity,apoptosis and expression of TP53 and BCL2 compared to the histologically identical lateral periodontal and gingival cysts

The botryoid odontogenic cyst (BOC) is a rare, locally more aggressive variant of the usually indolent lateral periodontal cyst (LPC) and gingival cyst (GC). A recent case of BOC provided an opportunity for an exploratory study on the causes of its more aggressive behavior. The limited objective was to see if the BOC was sufficiently different from the other cysts to warrant an investment in a large study. Sections of neutral buffered formalin fixed, paraffin-embedded tissues from the BOC and archival specimens of four GCs, four LPCs and three odontogenic keratocysts (OKCs) were stained using immunohistochemistry for Ki-67, a marker of proliferating cells, caspase-3, a marker of cells undergoing apoptosis, tumor suppressor p53, and the apoptosis inhibitor BCL2. The mean labeling index (LI) of immunoreactive cyst epithelial cells was computed for each antibody and type of cyst. Compared to the LPCs and GCs, the BOC exhibited a moderately larger Ki-67/caspase-3 LI difference, which indicates that the BOC had a net higher rate of growth. We found a much higher level of LI, therefore likely dysregulation of p53. We also found a much higher LI of BCL2. The LIs of p53 and BCL2 in the BOC were similar to and more than twice that of the OKCs, respectively. Although meaningful statistical analysis was precluded by our use of only one case of BOC and a small number of the other cysts, the high p53 and very high BCL2 labeling indices of the BOC offer a potential explanation for its reportedly more aggressive behavior that clearly is worthy of further investigation.     

New model for the formation and function of sagittocysts: Symsagittifera corsicae n. sp. (Acoela)

Abstract. This study is focused on the formation and function of sagittocysts, which are secretions typical of members of the acoel family Sagittiferidae. The needle-shaped sagittocysts are produced in specialized gland cells (sagittocytes) whose distal necks are often surrounded by muscle mantles. Contraction of the muscle mantle ejects the sagittocyst. We establish a model for the development of sagittocytes and muscle mantles out of the stem cell pool of the new acoel species Symsagittifera corsicae . We used various techniques, especially interference and phase-contrast microscopy of living specimens as well as labeling of the body-wall musculature, for species characterization. In addition to the morphological features, we provide the third complete sequence of the 18S rDNA gene in the family Sagittiferidae.     

Effects of high arsenic and fluoride soil concentrations on soybean plants

Arsenic (As) and Fluoride (F) are present in many soils, affecting crops and posing risks in the food chain. We performed pot experiments on spiked soils enriched in these elements either individually or simultaneously, over a wide range of concentrations. Soybean biomass production, grain yield, As and F accumulation and distribution within the plant, and the antioxidant response to these stresses were analyzed. Arsenic was more toxic than F. At As levels >35 mg/kg and F levels >375 mg/kg, yield loss reached 60% and 30%, respectively. At the highest dose of As plants died within 2 weeks, whereas F showed no lethality. When they were applied simultaneously, detrimental effects were more important. As and F in plants increased in all soybean organs although grains presented the lowest concentrations. Antioxidant enzymes were enhanced in plants but this increase was not high enough to cope with the oxidative damage.     

Taxol from fungal endophytes and the issue of biodiversity

Fungi represent one of the most understudied and diverse group of organisms. Commonly, these organisms make associations with higher life forms and may proceed to biochemically mimic the host organism. An excellent example of this is the anticancer drug, taxol, which had been previously supposed to occur only in the plant genusTaxus (yew). However, taxol has been reported in a novel endophytic fungus—Taxomyces andreanae, but also has been demonstrated to occur in a number of unrelated fungal endophytes includingPestalotia, Pestalotiopsis, Fusarium, Alternaria, Pithomyces, Monochaetia and others. Thus, this report presents information on the presence of taxol among disparate fungal genera, and uses these observations as an additional argument to support efforts to study fungal endophytes and preserve their associated host plants.     

Analysis of murine hepatitis virus strain A59 temperature-sensitive mutant TS-LA6 suggests that nsp10 plays a critical role in polyprotein processing

Coronaviruses are the largest RNA viruses, and their genomes encode replication machinery capable of efficient replication of both positive- and negative-strand viral RNAs as well as enzymes capable of processing large viral polyproteins into putative replication intermediates and mature proteins. A model described recently by Sawicki et al. (S. G. Sawicki, D. L. Sawicki, D. Younker, Y. Meyer, V. Thiel, H. Stokes, and S. G. Siddell, PLoS Pathog. 1:e39, 2005), based upon complementation studies of known temperature-sensitive (TS) mutants of murine hepatitis virus (MHV) strain A59, proposes that an intermediate comprised of nsp4 to nsp10/11 ( approximately 150 kDa) is involved in negative-strand synthesis. Furthermore, the mature forms of nsp4 to nsp10 are thought to serve as cofactors with other replicase proteins to assemble a larger replication complex specifically formed to transcribe positive-strand RNAs. In this study, we introduced a single-amino-acid change (nsp10:Q65E) associated with the TS-LA6 phenotype into nsp10 of the infectious clone of MHV. Growth kinetic studies demonstrated that this mutation was sufficient to generate the TS phenotype at permissive and nonpermissive temperatures. Our results demonstrate that the TS mutant variant of nsp10 inhibits the main protease, 3CLpro, blocking its function completely at the nonpermissive temperature. These results implicate nsp10 as being a critical factor in the activation of 3CLpro function. We discuss how these findings challenge the current hypothesis that nsp4 to nsp10/11 functions as a single cistron in negative-strand RNA synthesis and analyze recent complementation data in light of these new findings.     

Persistent Infection Promotes Cross-Species Transmissibility of Mouse Hepatitis Virus

Persistent infection with mouse hepatitis virus (MHV) strain A59 in murine DBT (delayed brain tumor) cells resulted in the emergence of host range variants, designated V51A and V51B, at 210 days postinfection. These host range mutants replicated efficiently in normally nonpermissive Chinese hamster ovary (CHO), in human hepatocarcinoma (HepG2), and to a lesser extent in human breast carcinoma (MCF7) cell lines. Little if any replication was noted in baby hamster kidney (BHK), green African monkey kidney (COS-7), feline kidney (CRFK), and swine testicular (ST) cell lines. By fluorescent antibody (FA) staining, persistent viruses V10B and V30B, isolated at days 38 and 119 days postinfection, also demonstrated very low levels of replication in human HepG2 cells. These data suggest that persistence may rapidly select for host range expansion of animal viruses. Pretreatment of HepG2 cells with a polyclonal antibody directed against human carcinoembryonic antigens (CEA) or with some monoclonal antibodies (Col-1, Col-4, Col-12, and Col-14) that bind human CEA significantly inhibited V51B infection. Under identical conditions, little or no blockade was evident with other monoclonal antibodies (kat4c or Col-6) which also bind the human CEA glycoproteins. In addition, an antibody (EDDA) directed against irrelevant antigens did not block V51B replication. Pretreatment with the Col-4 and Col-14 antibodies did not block Sindbis virus replication in HepG2 cells or MHV infection in DBT cells, suggesting that one or more CEA glycoproteins likely functioned as receptors for V51B entry into human cell lines. To test this hypothesis, the human biliary glycoprotein (Bgp) and CEA genes were cloned and expressed in normally nonpermissive BHK cell lines by using noncytopathic Sindbis virus replicons (pSinRep19). By growth curves and FA staining, human CEA and to a much lesser extent human Bgp functioned as receptors for V51B entry. Furthermore, V51B replication was blocked with polyclonal antiserum directed against human CEA and Bgp. Under identical conditions, the parental MHV strain A59 failed to replicate in BHK cells expressing human Bgp or CEA. These data suggest that MHV persistence may promote virus cross-species transmissibility by selecting for virus variants that recognize phylogenetic homologues of the normal receptor.