Determinants and effects of sinus worm Skrjabingylus nasicola (Nematoda: Metastrongyloidae) infestation in invasive American mink Neovison vison in Germany.

Skrjabingylus nasicola (Leuckart, 1842) are geographically widespread nematodes that parasitize the nasal and frontal sinus cavities of smaller mustelids. As most prior work was solely based on the analysis of bone injuries of museum skull, little is known about the determinants and effects of infestation in the host species. Working on fresh skulls, we aimed to analyze infestation patterns in American mink (Neovison vison Schreber, 1777) from nine study areas in northern Germany and to identify factors that explained infestation prevalence and intensity in the host species. The prevalence (46.7-62.9 %) and infestation intensity values (4.5-10.89 nematodes) reported here were relatively large, especially compared to other American mink populations in Europe. Considering mink diet, our study sites probably harbored a larger number of infested paratenic hosts and climate did not have a substantial negative influence on survival of S. nasicola larvae. We did not observe any significant sex-age differences in either prevalence or intensity of S. nasicola infestation. We did not find a negative impact of an infestation on the host animals' body weight, confirming prior results that the parasite is not a significant mortality factor in mustelids. Our study suggests that this holds even outside the native distributional range where the host's defenses might not be optimally adapted to an autochthonous parasite.

Spiers Memorial Lecture. Breeding and building molecular spies.

To circumvent the limited spatial resolution of fluorescent protein imaging, we are developing genetically encoded tags for electron microscopy (EM).

Comparative analysis of brain structure, metabolism, and cognition in myotonic dystrophy 1 and 2.

OBJECTIVE: Myotonic dystrophy type 1 and 2 (DM1/DM2) are multisystemic diseases with common cognitive deficits beside the cardinal muscular symptoms. We performed a comprehensive analysis of cerebral abnormalities to compare the neuropsychological defects with findings in different imaging methods in the same cohort of patients. METHODS: Neuropsychological investigations, structural cerebral MRI including brain parenchymal fraction (BPF) and voxel-based morphometry (VBM), and (18)F-deoxy-glucose PET (FDG-PET) were performed in patients (20 DM1 and 9 DM2) and matched healthy controls, and analyzed using statistical parametric mapping (SPM2). RESULTS: DM1 and DM2 patients showed typical neuropsychological deficits with a pronounced impairment of nonverbal episodic memory. Both patient groups showed a reduction of the global gray matter (measured by BPF), which could be localized to the frontal and parietal lobes by VBM. Interestingly, VBM revealed a bilateral hippocampal volume reduction that was correlated specifically to both a clinical score and episodic memory deficits. VBM also revealed a pronounced change of thalamic gray matter. White matter lesions were found in >50% of patients and their extent was correlated to psychomotor speed. FDG-PET revealed a frontotemporal hypometabolism, independent of the decrease in cortical gray matter. All abnormalities were similar in both patient groups but more pronounced for DM1. CONCLUSIONS: Our results suggest that 1) some of the characteristic cognitive deficits of these patients are linked to specific structural cerebral changes, 2) decreases in gray matter and metabolism are independent processes, and 3) the widespread brain abnormalities are more pronounced in DM1.

Recruitment of old genes to new functions: evidences obtained by comparing the orthologues of human XLMR genes in mouse and chicken.

Gene mapping data indicate that the human X chromosome is enriched in genes that affect both, higher cognitive efficiency and reproductive success. This raises the question whether these functions are ancient, or whether conserved X-linked genes were recruited to new functions. We have studied three X-linked mental retardation (XLMR) genes by RNA in situ hybridization in mouse and in chicken, in which these genes are autosomal: Rho guanine nucleotide exchange factor 6 (ARHGEF6), oligophrenin (OPHN1), and p21 activated kinase 3 (PAK3). In the mouse these genes are specifically expressed in telencephalic regions. Their orthologues in the chicken gave patterns of similar specificity in ancient parts of the brain, i.e. cerebellum and mesencephalon, but were not expressed in the telencephalon. Also in the testes, specific expression was only found in mouse, not in chicken. These data are interpreted such that certain genes on the X chromosome gained novel functions during evolution.

Fumarate hydratase enzyme activity in lymphoblastoid cells and fibroblasts of individuals in families with hereditary leiomyomatosis and renal cell cancer.

BACKGROUND: Hereditary leiomyomatosis and renal cell cancer (HLRCC) is the autosomal dominant heritable syndrome with predisposition to development of renal cell carcinoma and smooth muscle tumours of the skin and uterus. OBJECTIVE: To measure the fumarate hydratase (FH) enzyme activity in lymphoblastoid cell lines and fibroblast cell lines of individuals with HLRCC and other familial renal cancer syndromes. METHODS: FH enzyme activity was determined in the whole cell, cytosolic, and mitochondrial fractions in 50 lymphoblastoid and 16 fibroblast cell lines including cell lines from individuals with HLRCC with 16 different mutations. RESULTS: Lymphoblastoid cell lines (n = 20) and fibroblast cell lines (n = 11) from individuals with HLRCC had lower FH enzyme activity than cells from normal controls (p<0.05). The enzyme activity in lymphoblastoid cell lines from three individuals with mutations in R190 was not significantly different from individuals with other missense mutations. The cytosolic and mitochondrial FH activity of cell lines from individuals with HLRCC was reduced compared with those from control cell lines (p<0.05). There was no significant difference in enzyme activity between control cell lines (n = 4) and cell lines from affected individuals with other hereditary renal cancer syndromes (n = 22). CONCLUSIONS: FH enzyme activity testing provides a useful diagnostic method for confirmation of clinical diagnosis and screening of at-risk family members.

[5 years ofFusaria and mycotoxin monitoring - any use for practice?]. / 5 Jahre Fusarien-und Mykotoxinmonitoring in Mecklenburg-Vorpommern - ein Nutzen für die Praxis?

Since 1999 the harvest of grain in Mecklenburg-Vorpommern has been investigated yearly as to its Fusaria infection rate and contamination with mycotoxins. 376 samples, most of them winter wheat, were tested for the mycotoxins zearalenone and deoxynivalenol in 1999-2003. The investigations were carried out with an ELISA; some results were confirmed by a chromatographic method (HPLC). In 2002 the Fusaria infection rate was graded as moderate (17,4%), in the other years as low (max. 9%). Zearalenone was detected only in 2,7% of the samples (max.236 µg/kg). 9,6% of samples contained deoxynivalenol (max. 1,2 mg/kg). Therefore the mycotoxin contamination in grain, harvested in Mecklenburg-Vorpommern 1999-2003 and the risk for consumers and animals as well is thought to be low. These results should help the farmers to understand that mycotoxins are not necessarily the sole cause of depressions in yield and loss of animals. It should be possible to avoid a false interpretation of mycotoxin findings.

J3-crystallin of the jellyfish lens: similarity to saposins.

J3-crystallin, one of the three major eye-lens proteins of the cubomedusan jellyfish (Tripedalia cystophora), shows similarity to vertebrate saposins, which are multifunctional proteins that bridge lysosomal hydrolases to lipids and activate enzyme activity. Sequence alignment of deduced J3-crystallin indicates two saposin-like motifs arranged in tandem, each containing six cysteines characteristic of this protein family. The J3-crystallin cDNA encodes a putative precursor analogous to vertebrate prosaposins. The J3-crystallin gene has seven exons, with exons 2-4 encoding the protein. Exon 3 encodes a circularly permutated saposin motif, called a swaposin, found in plant aspartic proteases. J3-crystallin RNA was found in the cubomedusan lens, statocyst, in bands radiating from the pigmented region of the ocellus, in the tentacle tip by in situ hybridization, and in the embryo and larva by reverse transcription-PCR. Our data suggest a crystallin role for the multifunctional saposin protein family in the jellyfish lens. This finding extends the gene sharing evolutionary strategy for lens crystallins to the cnidarians and indicates that the putative primordial saposin/swaposin J3-crystallin reflects both the chaperone and enzyme connections of the vertebrate crystallins.

Characterization of the Saccharomyces cerevisiae homolog of the melatonin rhythm enzyme arylalkylamine N-acetyltransferase (EC 2.3.1.87).

Arylalkylamine N-acetyltransferase (AANAT, serotonin N-acetyltransferase, EC ) plays a unique transduction role in vertebrate physiology by converting information about day and night into a hormonal signal: melatonin. Only vertebrate members of the AANAT family have been functionally characterized. Here a putative AANAT from Saccharomyces cerevisiae (scAANAT) was studied to determine whether it possessed the catalytic activity of the vertebrate enzyme. scAANAT is 47% similar to ovine AANAT, but lacks the regulatory N- and C-terminal flanking regions conserved in all vertebrate AANATs. It was found to have enzyme activity generally typical for AANAT family members, although the substrate preference pattern was somewhat broader, the specific activity was lower, and the pH optimum was higher. Deletion of scAANAT reduced arylalkylamine acetylation by S. cerevisiae extracts, indicating that scAANAT contributes significantly to this process. The scAANAT sequence conformed to the three-dimensional structure of ovine AANAT catalytic core; however, an important structural element (loop 1) was found to be shorter and to lack a proline involved in substrate binding. These differences could explain the lower specific activity of scAANAT, because of the importance of loop 1 in catalysis. Data base analysis revealed the presence of putative AANATs in other fungi but not in the nearly complete genomes of Drosophila melanogaster or Caenorhabditis elegans. These studies indicate that the catalytic and kinetic characteristics of fungal and vertebrate enzymes can be considered to be generally similar, although some differences exist that appear to be linked to changes in one structural element. Perhaps the most striking difference is that fungal AANATs lack the regulatory domains of the vertebrate enzyme, which appear to be essential for the regulatory role the enzyme plays in photochemical transduction.

Demethylation, reactivation, and destabilization of human fragile X full-mutation alleles in mouse embryocarcinoma cells.

The major causes of fragile X syndrome are mutational expansion of the CGG repeat in the FMR1 gene, hypermethylation, and transcriptional silencing. Most fragile X embryos develop somatic mosaicism of disease-causing "full" expansions of different lengths. Homogeneity of the mosaic patterns among multiple tissues in the same individual indicates that these previously unstable expansions acquire mitotic stability early in fetal life. Since mitotic stability is found strictly associated with hypermethylation in adult tissues, current theory has fixed the time of instability to developmental stages when fully expanded CGG repeats exist in an unmethylated state. We used murine embryocarcinoma (EC) cells (PC13) as a model system of pluripotent embryonic cells. Hypermethylated and unmethylated full expansions on human fragile X chromosomes were transferred from murine A9 hybrids into EC cells, by means of microcell fusion. As demonstrated in the present study for the first time, even full expansion alleles that were fully methylated and stable in the donors' fibroblasts and in A9 became demethylated, reactivated, and destabilized in undifferentiated EC hybrids. When destabilized expansions were reintroduced from EC cells into A9, instability was reversed to stability. Our results strongly support the idea that fully expanded alleles are initially unstable and unmethylated in the human embryo and gain stability upon genetic or epigenetic change of the embryonic cells.

Human Ca(2+) receptor extracellular domain. Analysis of function of lobe I loop deletion mutants.

The G protein-coupled Ca(2+) receptor (CaR) possesses an approximately 600-residue extracellular domain involved in ligand binding and receptor activation. Based on an alignment of the amino acid sequence of the CaR with that of bacterial periplasmic-binding proteins, the first approximately 530 residues of the extracellular domain are believed to form a domain resembling a bilobed Venus's flytrap (VFT). Four insertions in the CaR sequence that do not align with those of bacterial periplasmic-binding proteins correspond to four loops within lobe I of the VFT. We constructed a series of deletion mutants of these four loops and tested their ability to form fully processed CaR as well as their ability to be activated by Ca(2+). As many as 21 residues (365) of loop III could be deleted without impairing receptor expression or activation. Deletion of portions of either loops I (50) or IV (438) did not impair receptor expression but significantly reduced Ca(2+) activation. Deletion of the entire loop II (117) abolished receptor expression and function, but the replacement of even a single residue within this deletion mutant led to expression of a monomeric form of the receptor showing increased Ca(2+) sensitivity but reduced maximal activation. Our results reveal that certain residues within loops I and IV are dispensable in formation of the VFT domain but are critical for Ca(2+) activation of the receptor. In contrast, the residues in loop II are critical for maintaining the inactive state of the CaR. We discuss these results in light of the recently defined crystal structure of the homologous domain of the type 1 metabotropic glutamate receptor.

[Not Available]. / Zur Bewertung von Mykotoxinbefunden in der Praxis.

Due to nonspecific clinical symptoms and the influence of different factors the diagnosis of mycotoxicosis is difficult in many cases. Case descriptions in dairy herds with chronic and acute diseases suspected of having a mycotoxicosis show the necessity of a solid differential diagnosis. Screening methods (MTT-assay) for mycotoxin analysis are recommended in this case. Systematic investigations of fodder cereals before feeding allow a prophylactical assessment of mycotoxin risk. In Mecklenburg-Vorpommern such investigations of the 1999 and 2000 harvest revealed only a low contamination with Zearalenon and Deoxynivalenol. Zearalenon was not detected in 133 samples. 31 of 157 samples contained DON in low levels.

Increase of FMRP expression, raised levels of FMR1 mRNA, and clonal selection in proliferating cells with unmethylated fragile X repeat expansions: a clue to the sex bias in the transmission of full mutations?

Fragile X syndrome is a triplet repeat disorder caused by expansions of a CGG repeat in the fragile X mental retardation gene (FMR1) to more than 220 triplets (full mutation) that usually coincide with hypermethylation and transcriptional silencing. The disease phenotype results from deficiency or loss of FMR1 protein (FMRP) and occurs in both sexes. The underlying full mutations arise exclusively on transmission from a mother who carries a premutation allele (60-200 CGGs). While the absolute requirement of female transmission could result from different mechanisms, current evidence favours selection or contraction processes acting at gametogenesis of pre- and full mutation males. To address these questions experimentally, we used a model system of cultured fibroblasts from a male who presented heterogeneous unmethylated expansions in the pre- and full mutation size range. On continual cell proliferation to 30 doublings we re-examined the behaviour of the expanded repeats on Southern blots and also determined the expression of the FMR1 gene by FMRP immunocytochemistry, western analysis, and RT-PCR. With increasing population doublings, expansion patterns changed and showed accumulation of shorter alleles. The FMRP levels were below normal but increased continuously while the cells that were immunoreactive for FMRP accumulated. The level of FMR1 mRNA was raised with even higher levels of mRNA measured at higher passages. Current results support the theory of a selection advantage of FMRP positive over FMRP deficient cells. During extensive proliferation of spermatogonia in fragile X males, this selection mechanism would eventually replace all full mutations by shorter alleles allowing more efficient FMRP translation. At the proliferation of oogonia of carrier females, the same mechanism would, in theory, favour transmission of any expanded FMR1 allele on inactive X chromosomes.

Omega -crystallin of the scallop lens. A dimeric aldehyde dehydrogenase class 1/2 enzyme-crystallin.

While many of the diverse crystallins of the transparent lens of vertebrates are related or identical to metabolic enzymes, much less is known about the lens crystallins of invertebrates. Here we investigate the complex eye of scallops. Electron microscopic inspection revealed that the anterior, single layered corneal epithelium overlying the cellular lens contains a regular array of microvilli that we propose might contribute to its optical properties. The sole crystallin of the scallop eye lens was found to be homologous to Omega-crystallin, a minor crystallin in cephalopods related to aldehyde dehydrogenase (ALDH) class 1/2. Scallop Omega-crystallin (officially designated ALDH1A9) is 55-56% identical to its cephalopod homologues, while it is 67 and 64% identical to human ALDH 2 and 1, respectively, and 61% identical to retinaldehyde dehydrogenase/eta-crystallin of elephant shrews. Like other enzyme-crystallins, scallop Omega-crystallin appears to be present in low amounts in non-ocular tissues. Within the scallop eye, immunofluorescence tests indicated that Omega-crystallin expression is confined to the lens and cornea. Although it has conserved the critical residues required for activity in other ALDHs and appears by homology modeling to have a structure very similar to human ALDH2, scallop Omega-crystallin was enzymatically inactive with diverse substrates and did not bind NAD or NADP. In contrast to mammalian ALDH1 and -2 and other cephalopod Omega-crystallins, which are tetrameric proteins, scallop Omega-crystallin is a dimeric protein. Thus, ALDH is the most diverse lens enzyme-crystallin identified so far, having been used as a lens crystallin in at least two classes of molluscs as well as elephant shrews.

Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements.

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5beta, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15N labels at the eta nitrogen positions of arginine side chains and 13C labels at glycine carbonyl positions and 13C-detected 13C-15N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5beta complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of phi and psi backbone dihedral angles in the RP135/0.53 complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13C-15N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.

Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca(2+) receptor critical for dimerization. Implications for function of monomeric Ca(2+) receptor.

We analyzed the effect of substituting serine for each of the 19 cysteine residues within the amino-terminal extracellular domain of the human Ca(2+) receptor on cell surface expression and receptor dimerization. C129S, C131S, C437S, C449S, and C482S were similar to wild type receptor; the other 14 cysteine to serine mutants were retained intracellularly. Four of these, C60S, C101S, C358S and C395S, were unable to dimerize. A C129S/C131S double mutant failed to dimerize but was unique in that the monomeric form expressed at the cell surface. Substitution of a cysteine for serine 132 within the C129S/C131S mutant restored receptor dimerization. Mutation of residues Cys-129, Cys-131, and Ser-132, singly and in various combinations caused a left shift in Ca(2+) response compared with wild type receptor. These results identify cysteines 129 and 131 as critical in formation of intermolecular disulfide bond(s) responsible for receptor dimerization. In a "venus flytrap" model of the receptor extracellular domain, Cys-129 and Cys-131 are located within a region protruding from one lobe of the flytrap. We suggest that this region represents a dimer interface for the receptor and that mutation of residues within the interface causes important changes in Ca(2+) response of the receptor.

Tissue-specific methylation differences in a fragile X premutation carrier.

Methylation of a premutation was found in a small percentage of blood cells in a male premutation carrier for the FMR1 mutation. To investigate the inter-tissue heterogeneity and possible clinical implications of this finding, fibroblast cells from the subject were also studied. Although the premutation size was found to be the same in leukocytes and fibroblasts, the methylation pattern was different. In cultured fibroblasts, the premutation was completely unmethylated, as is typical of premutations, whereas methylation of the premutation was detected in a small percentage of lymphocytes. However, the change in methylation did not affect the FMR1 protein (FMRP) expression, as immunocytochemical analysis of FMRP performed on cultured skin fibroblasts and a blood smear revealed normal levels of expression in both tissues.