Towards resolving and redefining Amphipyrinae (Lepidoptera,Noctuoidea, Noctuidae): a massively polyphyletic taxon

Amphipyrinae have long been a catchall taxon for Noctuidae, with most members lacking discernible morphological synapomorphies that would allow their assignment to one of the many readily diagnosable noctuid subfamilies. Here data from seven gene regions (> 5500 bp) for more than 120 noctuid genera are used to infer a phylogeny for Amphipyrinae and related subfamilies. Sequence data for 57 amphipyrine genera – most represented by the type species of the genus – are examined. We present here the first large‐scale molecular phylogenetic study of Amphipyrinae and the largest molecular phylogeny of Noctuidae to date; several proposed nomenclatural changes for well‐supported results; and the identification of areas of noctuid phylogeny where greater taxon sampling and/or genomic‐scale data are needed. Adult and larval morphology, along with life‐history traits, for taxonomic groupings most relevant to the results are discussed. Amphipyrinae are significantly redefined; many former amphipyrines, excluded as a result of these analyses, are reassigned to other noctuid subfamily‐level taxa. Four genera, Chamaeclea Grote, Heminocloa Barnes & Benjamin, Hemioslaria Barnes & Benjamin and Thurberiphaga Dyar, are transferred to the tribe Chamaecleini Keegan & Wagner tribe n. in Acontiinae. Stiriina is elevated to Stiriinae rev. stat. , Grotellina is elevated to Grotellinae rev. stat. and Annaphilina is elevated to Annaphilini rev. stat. Acopa Harvey is transferred to Bryophilinae, Aleptina Dyar is transferred to Condicinae, Leucocnemis Hampson and Oxycnemis gracillinea (Grote) are transferred to Oncocnemidinae, Nacopa Barnes & Benjamin is transferred to Noctuinae and Narthecophora Smith is transferred to Stiriinae. Azenia Grote (and its subtribe Azeniina), Cropia Walker, Metaponpneumata Möschler, Sexserrata Barnes & Benjamin and Tristyla Smith are transferred to Noctuidae incertae sedis. Hemigrotella Barnes & McDunnough (formerly in subtribe Grotellina) is retained in Amphipyrinae. Argentostiria Poole and Bistica Dyar are retained in Stiriini but removed from incertae sedis position. This published work has been registered on ZooBank: http://zoobank.org/urn:lsid:zoobank.org:pub:4A140782‐31BA‐445A‐B7BA‐6EAB98ED43FA .     

Modulation of differentiation markers in human choriocarcinoma cells by extracellular matrix: on the role of a three-dimensional matrix structure

Abstract. During spontaneous or chemically induced differentiation human choriocarcinoma cells express typical characteristics of the normal differentiating trophoblast: 1) increased production of peptide and steroid hormones (chorionic gonadotropin, placental lactogen, estrogens, progesterone); 2) increased activity of cellular alkaline phosphatase; 3) morphological transition from cytotrophoblast to syncytiotrophoblast-like cells; and 4) arrested cell proliferation. Since the extracellular matrix is known to control gene expression we have examined the effects of different substrates composed of matrix macromolecules on the differentiation of BeWo choriocarcinoma cells. Matrices tested were; fibronectin, laminin, collagens type I and type IV, the basement membrane-like complex matrix Matrigel, and a complex matrix extracted from human term placenta. Irrespective of the type of molecule(s), it was consistently found that, whenever the matrix molecules were presented as threedimensional structures (as opposed to protein coatings on tissue culture plastic) the response of affected differentiation markers monitored was highly pronounced. Morphology was changed from monolayers to rounded colonies, cell proliferation was reduced, and the secretion of chorionic gonadotropin was increased up to tenfold. Heterogeneous effects were observed on progesterone secretion and on the activity of cellular alkaline phosphatase. Cell adhesion to matrix molecules, however, did not depend on the structure of the matrix. This study demonstrates that gene expression in these tumor cells can be modified by extracellular matrix and highlights that not only the presence of effector molecules in the matrix but also the three-dimensional structure of the matrix is important for the induction of differentiation.     

Hereditary cystatin C amyloid angiopathy: identification of the disease-causing mutation and specific diagnosis by polymerase chain reaction based analysis

Summary Hereditary cystatin C amyloid angiopathy (HCCAA) is a dominantly inherited disease characterized by amyloidosis, dementia and fatal cerebral hemorrhage of young adults. A method for rapid and simple diagnosis of HCCAA is described. It is based upon oligonucleotide-directed enzymatic amplification of a 275-bp genomic DNA segment containing exon 2 of the cystatin C gene from a blood sample, followed by digestion of the amplification product with AluI. Loss of an AluI recognition site in the amplified DNA segment from HCCAA patients results in a deviating band-pattern at agarose gel electrophoresis, compared with that obtained from normal subjects or unaffected HCCAA family members. In a population of 9 patients with manifest HCCAA, 14 patients with other causes of brain hemorrhage and 16 healthy individuals, the diagnostic procedure displayed a sensitivity and specificity for HCCAA of 100%. Amplified DNA segments from 4 HCCAA patients of four different families were analyzed by nucleotide sequencing; the HCCAA-causing mutation in all families was found to be a single TA substitution in the codon for amino acid residue 68 of cystatin C.     

Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus

Summary Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12–13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.     

Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E

Summary A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 µg. Parallel control groups received analogous inoculations of an isotype-matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer-sensitive rat T cell lymphoma G1—Tc1. The cytotoxic cell population was more than 90% CD4⁺. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals.     

Changes in haemolymph ion concentrations of Astacus astacus L. and Pacifastacus leniusculus (Dana) after exposure to low pH and aluminium

During exposure to soft water, acidified to pH 4.0, the haemolymph concentrations of Na⁺, K⁺, and Cl^– decreased whereas the Ca²⁺ concentration fluctuated in Astacus astacus. The haemocyte content of K⁺ decreased from 9% to 2% of the total haemolymph K⁺ content after exposure to pH 3.7 for 3 days. Within 14 days, 250 µg Al³⁺ l^–1, as Al₂(SO₄)₃ at pH 5.0, reduced the haemolymph Na⁺ content in Astacus astacus and Pacifastacus leniusculus, however, the effects were less pronounced than earlier reported for fish. Disturbed ion regulation, mainly depending on low pH, is thought to contribute to the absence of these species in acid waters.     

Distribution of sulphate and iduronic acid residues in heparin and heparan sulphate

1. A method was developed for determination of the uronic acid composition of heparin-like glycosaminoglycans. Polymers or oligosaccharides are degraded to monosaccharides by a combination of acid hydrolysis and deamination with HNO₂. The resulting uronic acid monosaccharides (accounting for about 70% of the uronic acid contents of the starting materials) are isolated and converted into the corresponding aldono-1,4-lactones, which are separated by g.l.c. The calculated ratios of glucuronic acid/iduronic acid are reproducible within 5%. 2. Samples of heparin from pig intestinal mucosa (molar ratio of sulphate/disaccharide unit, 2.40) and heparan sulphate from human aorta (sulphate/disaccharide ratio, 0.46) were subjected to uronic acid analysis. l-Iduronic acid constituted 77% and 19% respectively of the total uronic acid contents. 3. The correlation between the contents of sulphate and iduronic acid indicated by this finding also applied to the fractionated deamination products of the two polymers. The sulphated fragments varied in size from disaccharide to octasaccharide (or larger) and showed sulphate/disaccharide molar ratios in the range of 0.05–2.0. The proportion of iduronic acid increased with increasing ester sulphate contents of the oligosaccharides. 4. Previous studies on the biosynthesis of heparin in a cell-free system have shown that l-iduronic acid residues are formed by C-5 epimerization of d-glucuronic acid units at the polymer level; the process requires concomitant sulphation of the polymer. The results obtained in the present structural study conform to these findings, and suggest further that similar mechanisms may operate in the biosynthesis of heparan sulphate. The epimerization reaction appears to be linked to the sulphation of hydroxyl groups but does not seem to require sulphation of the target uronic acid residues. The significance of sulphamino groups in relation to the formation of iduronic acid is unknown.     

Spike protein oligomerization control of Semliki Forest virus fusion.

We have recently shown, using cleavage-deficient mutants of the p62-E1 membrane protein complex of Semliki Forest virus that p62 cleavage to E2 is necessary for the activation of the fusion function of the complex at pH 5.8 (a pH optimal for virus fusion) (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990). In this study, we show that the mutant precursor complexes can be induced to activate membrane fusion when treated with more acidic buffers (pH 5.0 and 4.5), which also appear to dissociate most of the p62-E1 complexes and change the conformation of the E1 subunit (the supposed fusion protein of Semliki Forest virus into a form which is resistant to trypsin digestion. These data suggest that p62 cleavage is not essential for membrane fusion per se but that the crucial event activating this process seems to be the apparent dissociation of the heterodimer, which in turn is facilitated by the spike precursor cleavage.     

The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy,is located on chromosome 20

Summary Hereditary cystatin C amyloid angiopathy has recently been shown to be caused by a point mutation in the cystatin C gene. To determine the chromosomal localization of the gene, 20 human-rodent somatic cell hybrids and a fulllength cystatin C cDNA probe were used. Southern blot analysis of BamHI digested cell hybrid DNA revealed that the probe recognizes a 10.6 kb human specific fragment and that this fragment cosegregates with human chromosome 20. Therefore, the human cystatin C gene (CST3) was assigned to chromosome 20.     

Rapid colorimetric detection of in vitro amplified DNA sequences

A colorimetric assay to detect immobilized amplified nucleic acids has been designed. This approach provides a rapid assay, suitable for clinical diagnosis, to analyze DNA sequences amplified by the polymerase chain reaction. The specific DNA sequences are captured on a solid support by the use of a recombinant fusion protein consisting of the Escherichia coli lac repressor and staphylococcal protein A. The biotin streptavidin system is used to detect the immobilized material. Positive samples can be analyzed by direct solid-phase sequencing. Here, we show that this nonradioactive concept can be used for analysis of Staphylococci and Streptococci and for specific detection of the protozoa Plasmodium falciparum in clinical samples.