Carapace dentition patterns, morphometrics and allozyme differentiation amongst two toothed freshwater crab species (Potamonautes warreni and P-unispinus) (Decapoda : Brachyura : Potamonautidae) from river systems in South Africa

The taxonomic relationship between two toothed South African river crabs, Potamonautes warreni and P. unispinus, is unclear. The problem stems from the widespread variation in carapace dentition patterns amongst P. warreni individuals over its biogeographic range, where single toothed individuals may appear similar in carapace morphology to P. unispinus. Ten populations of P. warreni and 18 populations of P. unispinus were collected and the morphometric and genetic differentiation between the two taxa quantified. Patterns of morphometric and genetic variation were examined using multivariate statistics and protein gel electrophoresis, respectively. Principal component analyses of carapace characters showed that the two species are morphologically indistinguishable. However, discriminate functions analyses and additional statistical results corroborate the morphological distinction between the two taxa. Allozyme electrophoresis of 17 protein coding loci, indicated a close genetic similarity between the two species (I = 0.92). A fixed allelic difference at one locus (LT-2) and extensive genetic variability at another locus (PGM-1) indicate that two gene pools are present and that the two taxa are genetically isolated. Intraspecific genetic I values for both species were > 0.97 and indicated no apparent genetic structuring on a micro or macro-geographic scale. The variation in carapace dentition among P. warreni populations possesses no genetic basis and may possibly toe the product of ecogenesis. The value of dentition patterns in the systematics of river crabs is discussed. Dentition patterns among river crab species appear to be conserved and reliable as species specific diagnostic markers, but should ideally be used in combination with other morphological data sets and genetic evidence.

DAF yields a cloned marker linked to the soybean (Glycine max) supernodulation nts-1 locus

We report a further characterization of the genomic region containing the soybean supernodulation gene NTS-1. We performed a search for new markers linked to NTS-1 by combining DNA amplification fingerprinting (DAF) and bulked segregant analysis (BSA). The search resulted in one cloned polymorphism (B44-456) linked in trans, 8.5cM from the locus. Southern hybridization showed duplication of the B44-456 sequence in the soybean genome. Additionally, a DNA database search revealed one Arabidopsis thaliana genomic clone from chromosome I possessing 62% homology to the B44-456 marker. A relatively low number of polymorphisms were identified by several PCR marker technologies for this soybean genomic region, providing an additional support for its highly conserved and/or duplicated organization.

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Design of a polyepitope construct for the induction of HLA-A0201-restricted HIV 1-specific CTL responses using HLA-A*0201 transgenic, H-2 class IKO mice

HLA-A*0201 transgenic, H-2D(b)/mouse beta2-microglobulin double-knockout mice were used to compare and optimize the immunogenic potential of 17HIV 1-derived, HLA-A0201-restricted epitopic peptides. A tyrosine substitution in position 1 of the epitopic peptides, which increases both their affinity for and their HLA-A0201 molecule stabilizing capacity, was introduced in a significant proportion, having verified that such modifications enhance their immunogenicity in respect of their natural antigenicity. Based on these results, a 13-polyepitope construct was inserted in the pre-S2 segment of the hepatitis B middle glycoprotein and used for DNA immunization. Long-lasting CTL responses against most of the inserted epitopes could be elicited simultaneously in a single animal with cross-recognition in several cases of their most common natural variants.

Cytotoxic T cell polyepitope vaccines delivered by ISCOMs

CD8 alpha beta cytotoxic T lymphocyte (CTL) polyepitope or polytope vaccines have traditionally been delivered using recombinant vector or DNA based delivery modalities. Here we show the delivery of polytope vaccines in the form of either synthetic polypeptides or recombinant polytope proteins by ImmunoStimulatory COMplexes (ISCOMs (R)). Induction of multiple protective CTL responses by these polytope-ISCOM formulations were comparable to viral vector or DNA based delivery modalities as assessed by IFN gamma ELISpot, chromium release and viral challenge assays. Measurement of CTL responses specific for the different epitopes revealed imunodominance patterns, which were largely independent of the vaccine vector or the order of the epitopes in the polytope. ISCOMs thus emerge as a viable human delivery modality for protein-based polytope vaccines. (C) 2001 Elsevier Science Ltd. All rights reserved.

Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have earned this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box, Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes. (C) 2001 Elsevier Science B.V. All rights reserved.

Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with G beta

There is increasing evidence that heterotrimeric G-proteins (G-proteins) are involved in many plant processes including phytohormone response, pathogen defence and stomatal control. In animal systems, each of the three G-protein subunits belong to large multigene families; however, few subunits have been isolated from plants. Here we report the cloning of a second plant G-protein γ-subunit (AGG2) from Arabidopsis thaliana. The predicted AGG2 protein sequence shows 48% identity to the first identified Arabidopsis Gγ-subunit, AGG1. Furthermore, AGG2 contains all of the conserved characteristics of γ-subunits including a small size (100 amino acids, 11.1 kDa), C-terminal CAAX box and a N-terminal α-helix region capable of forming a coiled-coil interaction with the β-subunit. A strong interaction between AGG2 and both the tobacco (TGB1) and Arabidopsis (AGB1) β-subunits was observed in vivo using the yeast two-hybrid system. The strong association between AGG2 and AGB1 was confirmed in vitro. Southern and Northern analyses showed that AGG2 is a single copy gene in Arabidopsis producing two transcripts that are present in all tissues tested. The isolation of a second γ-subunit from A. thaliana indicates that plant G-proteins, like their mammalian counterparts, may form different heterotrimer combinations that presumably regulate multiple signal transduction pathways.

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface, We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodo- main of the interleukin-2 alpha (IL-2 alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK1 epithelial cells, Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin, Truncation mutants unable to bind beta -catenin were correctly targeted, showing, contrary to current understanding, that beta -catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine mediated targeting is maintained in UC-PK, cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line, These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

MUC1: Antibodies and immunoassays

High molecular weight mucins represent a unique challenge as tumor markers by virtue of their complex array of epitopes, The list is dominated by the high molecular weight mucins MUC1, CEA and CA125. While the currently accepted role for these tumor markers is in the prediction and detection of relapse, it is possible that their sensitivity and specificity can be improved. Although immunoassays detecting the tumor marker MUC1 are both sensitive and specific for predicting relapse in breast cancer, so far they are not in widespread use in the follow-up of this disease. Are there new combinations of conventional reagents that could improve assay sensitivity, or should we be looking for more radical changes in assay design incorporating combinatorial technology? Copyright (C) 2001 S. Karger AG, Basel.

neptune, a Kruppel-like transcription factor that participates in primitive erythropoiesis in Xenopus

The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Kruppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates beta -globin expression during erythroid differentiation [2-6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptune's DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus.

The nutritive value of Rhodes grass (Chloris gayana) when treated with CaO, NaOH or a microbial inoculant and offered to dairy heifers as big-bale silage

A series of laboratory and animal studies examined the use of chemical and biological agents to enhance the digestibility of Rhodes grass (grass) cut at 60 (young) and 100 (mature) days of regrowth and ensiled as big round bales. The treatments included an untreated control (C), a microbial inoculant (I), NaOH, CaO and NaOH plus inoculant (NaOH + I). Inoculant was grown anaerobically, using a starter culture of rumen fluid from cattle given Rhodes grass. Treatments C, 1, NaOH, NaOH + I, were offered separately to twelve dairy heifers, in a 3 X 4 randomized complete block design, repeated twice for each grass silage. C and I had substantial mould growth, compared with no visible mould in NaOH or NaOH + 1. CaO treatment was effective in preventing mould growth, but had little effect on the chemical composition and in sacco digestibility of mature grass silage. NaOH reduced NDF content and increased in sacco digestibility (P < 0.05) but not the in vivo digestibility (P > 0.05) of both mature- and young-grass silage. The effects of other treatments on nutritive value were non-significant at both stages of maturity. NaOH increased the intake of mature-grass silage by 24-26% (P < 0.05), but had little effect on the intake of young-grass silage (P > 0.05). Treatment I consistently reduced grass silage intake (P < 005) for young-grass silage. The findings of these studies show that treating mature Rhodes grass with NaOH will improve its nutritive value and reduce mould growth in conserved herbage. However none of the treatments in this study had any consistently positive effects on the in vivo nutritive value or storage quality of young-grass silage.

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold MP1 on a late endosomal/lysosomal compartment

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro. The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Cloning and functional analysis of the Sry-related HMG box gene, Sox18

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii);ln intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor-2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue. (C) 2001 Elsevier Science B.V. All rights reserved.

Integrability and exact spectrum of a pairing model for nucleons

A pairing model for nucleons, introduced by Richardson in 1966, which describes proton-neutron pairing as well as proton-proton and neutron-neutron pairing, is re-examined in the context of the quantum inverse scattering method. Specifically, this shows that the model is integrable by enabling the explicit construction of the conserved operators. We determine the eigenvalues of these operators in terms of the Bethe ansatz, which in turn leads to an expression for the energy eigenvalues of the Hamiltonian.

Exact solution at integrable coupling of a model for the Josephson effect between small metallic grains

A model is introduced for two reduced BCS systems which are coupled through the transfer of Cooper pairs between the systems. The model may thus be used in the analysis of the Josephson effect arising from pair tunneling between two strongly coupled small metallic grains. At a particular coupling strength the model is integrable and explicit results are derived for the energy spectrum, conserved operators, integrals of motion, and wave function scalar products. It is also shown that form factors can be obtained for the calculation of correlation functions. Furthermore, a connection with perturbed conformal field theory is made.