Cadmium and zinc induction of ZnT-1 mRNA in an established carp cell line

The cDNA of a zinc transporter-1 (ZnT-1) gene was cloned from an established cell line derived from common carp (Cyprinus carpio) using rapid amplification of cDNA ends (RACE). Using real-time quantitative PCR, we showed that both zinc (Zn) and cadmium (Cd) transiently upregulate ZnT-1 mRNA to comparable levels. The loosely bound cellular Zn pool, as estimated using the Zn-specific probe FluoZin-3, was increased threefold after exposure to 250 microM ZnCl(2). Correspondingly, the ZnT-1 mRNA level at 24 h was induced about fivefold, reflecting the need for more zinc export capacity. Total cellular zinc levels were not different from the controls after 72 h of exposure to 10, 50, or 250 microM ZnCl(2). A loss of total cellular Zn but little labile zinc changes were observed with up to 25 microM Cd. At 72 h, the total Zn was partially restored to the control levels, only 1 microM Cd allowed for a full recovery. Downregulation of ZnT-1 mRNA and partial loss of loosely bound Zn were observed with 50 microM Cd. Our results clearly show that although Zn and Cd can both regulate Zn export in EPC cells, the effects on the cellular Zn pools are quite different.     

A Comparison of Population Size Estimators under the Truncated Count Model With and Without Allowance for Contaminations

The purpose of the study is to estimate the population size under a homogeneous truncated count model and under model contaminations via the Horvitz‐Thompson approach on the basis of a count capture‐recapture experiment. The proposed estimator is based on a mixture of zero‐truncated Poisson distributions. The benefit of using the proposed model is statistical inference of the long‐tailed or skewed distributions and the concavity of the likelihood function with strong results available on the nonparametric maximum likelihood estimator (NPMLE). The results of comparisons, for finding the appropriate estimator among McKendrick's, Mantel‐Haenszel's, Zelterman's, Chao's, the maximum likelihood, and the proposed methods in a simulation study, reveal that under model contaminations the proposed estimator provides the best choice according to its smallest bias and smallest mean square error for a situation of sufficiently large population sizes and the further results show that the proposed estimator performs well even for a homogeneous situation. The empirical examples, containing the cholera epidemic in India based on homogeneity and the heroin user data in Bangkok 2002 based on heterogeneity, are fitted with an excellent goodness‐of‐fit of the models and the confidence interval estimations may also be of considerable interest. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A redescription of some poorly known Rotliegend arachnids from the Lower Permian (Asselian) of the Ilfeld and Thuringian Forest Basins, Germany

Two poorly known trigonotarbids (Arachnida: Trigonotarbida),Eophrynus schärft Scharf, 1924 andEophrynus ilfeldicus Scharf, 1924 from the Lower Permian (Asselian) Ilfeld Basin in the Harz Region of Germany are redescribed. The latter is transferred to the genusAphantomartus Pocock, 1911. The former resembles members of Lissomartidae, but given its incompleteness it is assigned here to Trigonotarbida incertae sedis. A phalangiotarbid (Arachnida: Phalangiotarbida) from Ilfeld described asOpiliotarbus elongatus (Scudder, 1890) represents the youngest record of this group. With respect to the genusOpiliotarbus Pocock, 1910,Architarbus hoffmanni Guthörl, 1934 — one of three names erected for a lost specimen from the Upper Carboniferous of the Saar region of Germany — represents the senior synonym ofOpiliotarbus kliveri Waterlot, 1934 andGoniotarbus sararm Guthörl, 1965. The Lower Permian fossilRhabdotarachnoides simoni Haupt, 1957 from the Rotliegend of the Thuringian Forest Basin, Germany was described as an arachnid and tentatively referred to Opiliones. Since it is identified here as a coalified plant remain, this species is regarded as nomen dubium.     

First detection of a chloroperoxidase in bryophytes

Chlorinated cyclic bisbibenzyls of the isoplagiochin type are the first verified halometabolites from bryophytes. They could be obtained by in vitro chlorination of isoplagiochin C with chloroperoxidase from Caldariomyces fumago. Furthermore, an enzyme of this type was detected for the first time in bryophytes namely in the liverwort Bazzania trilobata using the monochlorodimedon assay.     

The use of RAPD in ecotoxicology

Toxic compounds may interfere with the genetic constitution of populations, either directly through mutagenic activity, or indirectly via population-mediated processes (i.e. selection, bottleneck). These processes are initiated when toxic compounds reduce the survival and/or fecundity of exposed organisms, either through the accumulation of unfavorable mutations or when they adversely affect the physiology of an organism and/or the environment in which it has to survive. In this review, we describe how the RAPD technique can be applied in an ecotoxicological context, providing information on all direct and indirect routes through which toxicants may affect the genetic structure of populations. Based on RAPD band intensity, gain/loss and band numbers, three major types of RAPD fingerprint analyses are discussed, yielding diagnostic, phenetic and genetic information. Ecotoxicological literature examples demonstrate that, under strictly standardized conditions, the RAPD technique can be a useful tool to preliminary assess toxicological population genetic effects, particularly since this technique is relatively inexpensive and yields information on a large number of loci without having to obtain sequence data for primer design. However, currently only a small fraction of its potential is used in ecotoxicology. Statistical tools and parameters, as used in other RAPD studies, should be applied in ecotoxicological research as well in order to fully exploit the potential of this technique. Finally, due to their random nature, RAPD data often must be considered as preliminary until they are further documented by cloning, sequencing and probing techniques.     

Single-vector three-frame expression systems for affinity-tagged proteins

An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.     

BRCA1 supports XIST RNA concentration on the inactive X chromosome

BRCA1, a breast and ovarian tumor suppressor, colocalizes with markers of the inactive X chromosome (Xi) on Xi in female somatic cells and associates with XIST RNA, as detected by chromatin immunoprecipitation. Breast and ovarian carcinoma cells lacking BRCA1 show evidence of defects in Xi chromatin structure. Reconstitution of BRCA1-deficient cells with wt BRCA1 led to the appearance of focal XIST RNA staining without altering XIST abundance. Inhibiting BRCA1 synthesis in a suitable reporter line led to increased expression of an otherwise silenced Xi-located GFP transgene. These observations suggest that loss of BRCA1 in female cells may lead to Xi perturbation and destabilization of its silenced state.     

Neuronal differentiation is accompanied by NSP-C expression

Neuroendocrine-specific protein (NSP) reticulons are expressed in neural and neuroendocrine tissues and cell cultures derived therefrom, while most other cell types lack NSP-reticulons. Three major subtypes have been identified so far, designated NSP-A, NSP-B, and NSP-C. We have investigated the correlation between the degree of neuronal differentiation, determined by morphological and biochemical criteria, and NSP-reticulon subtype expression. For this purpose, several human neuroblastoma cell lines, exhibiting different degrees of neuronal differentiation, were examined immuno(cyto) chemically. It became obvious that the expression of NSP-C, as detected by immunofluorescence microscopy and Western blotting, is most prominent in cell lines with a high degree of neuronal differentiation, such as LA-N-5. Such highly differentiated cells also express other neural and neuroendocrine markers, such as neural cell adhesion molecule (NCAM), neurofilament proteins, synaptophysin, and chromogranin. NSP-A was observed in all cell lines to a different extent. However, no clear correlation was observed with the degree of neuronal differentiation as defined by other neuronal and neuroendocrine markers or morphology. NSP-B could not be detected. The induction of neuronal differentiation with nerve growth factor, dbcAMP, and retinoic acid in the rat pheochromocytoma cell line PC12 and the human teratocarcinoma cell line hNT2, respectively, induced the expression of NSP-A and NSP-C in these cell lines parallel to the induction of neurofilament protein expression. It is concluded that NSP-C expression, in particular, is strongly correlated with neuronal differentiation.