Double coupling Edman chemistry for high-sensitivity automated protein sequencing

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a promising new method for the analysis of protein sequencing products. It gives 10 zmol (1 zmol = 10(-21) mol) limits of detection (3 sigma) for fluorescein thiohydantoin (FTH) amino acids. We have developed a separation for the (FTH)-amino acid products generated from 18 of the 20 coded amino acids. The extremely low volume requirement associated with CE-LIF makes it incompatible with commercial sequencers. For this reason, we have also been developing a miniaturized sequencer that can be more easily coupled to our detection system. Both the CE-LIF system and the miniaturized sequencer are described.     

A combinatorial phage display library for the generation of specific Fab fragments recognizing human spermatozoa and inhibiting fertilizing capacity in vitro

To select a source of lymphocytes for the generation of an anti-sperm-biased combinatorial phage display library, venous blood was obtained from 34 vasovasostomy (vasectomy reversal) patients approximately 3 mo after surgery. Using a variety of immunoassays, serum was analyzed for antibodies against human spermatozoa, and a patient was selected on the basis of high titer of antibodies that recognized the equatorial region of the sperm head and inhibited sperm fertilizing capacity in vitro. Total RNA isolated from the stored lymphocytes of this individual was reversed transcribed, and gamma1 (Fd) region and kappa chains were amplified by polymerase chain reaction for the successful construction of an antibody phage display library. The library was panned against human spermatozoa to isolate sperm-specific phage that recognized the equatorial region of the sperm head. Three preparations of Fab were tested via the hamster egg penetration test. Each preparation significantly (p < 0. 005) inhibited sperm-egg binding and fusion, with one preparation (designated Fab-G) causing complete inhibition. Sequence analysis of the kappa light gene encoding Fab-G revealed a 93% homology with the light chain of human anti-human immunodeficiency virus gp120 p35 variable region. This technology may have a practical application in characterization of the immune response to spermatozoa and for the design of sperm-based contraceptive vaccines.     

The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1

Toxoplasma gondii is an Apicomplexan parasite with a complex life cycle that includes a rapidly dividing asexual stage known as the tachyzoite. The tachyzoite surface has been reported to comprise five major antigens, the most abundant of which is designated SAG1 (for surface antigen 1). At least one of the other four (SAG3) and another recently described minor antigen (SRS1 [for SAG1-related sequence 1]) have previously been shown to be structurally related to SAG1. To determine if further SAG1 homologs exist, we searched a Toxoplasma expressed sequence tag (EST) database and found numerous ESTs corresponding to at least three new genes related to SAG1. Like SAG1, these new SRS genes encode apparently glycosylphosphatidylinositol-anchored proteins that share several motifs and a set of conserved cysteine residues. This family appears to have arisen by divergence from a common ancestor under selection for the conservation of overall topology. The products of two of these new genes (SRS2 and SRS3) are shown to be expressed on the surface of Toxoplasma tachyzoites by immunofluorescence. We also identified strain-specific differences in relative expression levels. A total of 10 members of the SAG1 gene family have now been identified, which apparently include three of the five major surface antigens previously described and one antigen expressed only in bradyzoites. The function of this family may be to provide a redundant system of receptors for interaction with host cells and/or to direct the immune responses that limit acute T. gondii infections.     

Regulation of BRCA1 and BRCA2 expression in human breast cancer cells by DNA-damaging agents

Germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 have been linked to the development of breast cancer, ovarian cancer, and other malignancies. Recent studies suggest that the BRCA1 and BRCA2 gene products may function in the sensing and/or repair of DNA damage. To investigate this possibility, we determined the effects of various DNA-damaging agents and other cytotoxic agents on the mRNA levels of BRCA1 and BRCA2 in the MCF-7 and other human breast cancer cell lines. We found that several agents, including adriamycin (a DNA intercalator and inhibitor of topoisomerase II), camptothecin (a topoisomerase I inhibitor), and ultraviolet radiation induced significant decreases in BRCA1 and BRCA2 mRNA levels. Decreased levels of BRCA1 and BRCA2 mRNAs were observed within 6-12 h after treatment with adriamycin and persisted for at least 72 h. Adriamycin also induced decreases in BRCA1 protein levels; but these decreases required several days. U.V. radiation induced dose-dependent down-regulation of BRCA1 and BRCA2 mRNAs, with significant decreases in both mRNAs at doses as low as 2.5 J/m2, a dose that yielded very little cytotoxicity. Adriamycin-induced down-regulation of BRCA1 and BRCA2 mRNAs was first observed at doses that yielded relatively little cytotoxicity and little or no apoptotic DNA fragmentation. Adriamycin and U.V. radiation induced distinct dose- and time-dependent alterations in the cell cycle distribution; but these alterations did not correlate well with corresponding changes in BRCA1 and BRCA2 mRNA levels. However, the adriamycin-induced reduction in BRCA1 and BRCA2 mRNA levels was correlated with p53 functional status. MCF-7 cells transfected with a dominant negative mutant p53 (143 val-->ala) required at least tenfold higher doses of adriamycin to down-regulate BRCA1 and BRCA2 mRNAs than did parental MCF-7 cells or control-transfected MCF-7 clones. These results suggest that BRCA1 and BRCA2 may play roles in the cellular response to DNA-damaging agents and that there may be a p53-sensitive component to the regulation of BRCA1 and BRCA2 mRNA expression.     

Diversity "down under": monogeneans in the Antipodes (Australia) with a prediction of monogenean biodiversity worldwide

There are approximately 25,000 species of fishes known in the world. The Monogenea are believed to be among the most host-specific of parasites and if each species of fish is host to a different species of monogenean, there could be almost 25,000 monogenean species on Earth. Currently, I estimate that between 3000 and 4000 of these are described. Australia has a rich marine fish fauna with approximately 3500 species of teleosts. If the same formula of one monogenean species per host fish species is applied, Australia marine fishes could host potentially 3500 species of monogeneans. The first monogenean species described from Australia was Encotyllabe pagrosomi MacCallum, 1917 and approximately 300 more species have since been described from the continent. Even in a region of Australia such as Heron Island on the Great Barrier Reef that has been a focus of sustained research on these parasites, only about 85 species are described from 40 of the most common, easily-caught species of fish. Reasons are discussed for the relatively small numbers of monogenean species described so far from Australia. Endemicity is difficult to judge, but only one is certain: Concinnocotyla australensis (Polystomatidae) from Neoceratodus forsteri (Dipnoi). Despite reductions in research funding, the value of parasite taxonomy must not be underestimated, particularly in regions of the world that have a rich diversity of potential hosts.     

Insulin-stimulated cell growth in insulin receptor substrate-1-deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase-independent pathway

The projections of enteric neurons to the circular muscle of the guinea pig gastric corpus were investigated systematically by using the retrogradely transported fluorescent carbocyanine dye 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI), applied to the muscle layer or myenteric plexus in vitro. DiI-labeled motor neuron cell bodies were located up to 6.3 mm aboral, 17 mm oral, and up to 20 mm circumferential to the DiI application site. Labeled nerve fibers ran for long distances from the DiI application site toward the greater and lesser curvatures, where they coursed parallel to the bundles of the "gastric sling" muscle. The majority of labeled cells were located toward the lesser curvature of the stomach. Nerve cell bodies that were aboral to the DiI application site were usually small, immunoreactive for choline acetyltransferase, and, thus, were likely to be excitatory motor neurons. Neurons that were located orally were larger, fewer in number, and immunoreactive for nitric oxide synthase and, thus, were likely to be inhibitory motor neurons. Application of DiI directly to the myenteric plexus filled neurons up to 15 mm aborally and up to 21 mm orally but labeled few neurons circumferentially. All nerve cells that were filled from either the circular muscle or the myenteric plexus had Dogiel type I morphological features. These results demonstrate a clear polarity of projection of inhibitory and excitatory motor neurons and a functionally continuous innervation of the circular and gastric sling muscle layers. Nonmotor neurons in the myenteric plexus were demonstrated, but neurons with Dogiel type II morphological features are apparently absent.     

Sentinel node localization in patients with breast cancer

BACKGROUND: Intraoperative lymphatic mapping and identification of the first draining lymph node (the sentinel node) may allow some patients with breast cancer to avoid the morbidity of formal axillary clearance. The aim of this pilot study was to establish the reliability of the technique in predicting axillary node status. METHODS: Sixty-eight consecutive patients with breast cancer, 38 undergoing mastectomy and 30 wide local excision, were included. Some 2-4 ml of 2.5 per cent Patent Blue dye was injected into adjacent breast tissue on the axillary side of the primary tumour. After 5-10 min, the axilla was explored. Blue-stained lymphatics were dissected to the sentinel node, which was removed for frozen-section examination, followed by routine histology. Formal axillary dissection was then completed. RESULTS: A sentinel lymph node was identified successfully in 56 (82 per cent) of 68 patients. Histology of the sentinel node accurately predicted axillary node status in 53 (95 per cent). There were three false negatives (5 per cent). In each case, only a single non-sentinel node was tumour positive. Sensitivity and specificity were 83 and 100 per cent respectively. CONCLUSION: This technique would allow a selective policy of formal axillary dissection in only node-positive patients; however, further experience and refinement are needed.     

The pore-lining region of shaker voltage-gated potassium channels: comparison of beta-barrel and alpha-helix bundle models

Although there is a large body of site-directed mutagenesis data that identify the pore-lining sequence of the voltage-gated potassium channel, the structure of this region remains unknown. We have interpreted the available biochemical data as a set of topological and orientational restraints and employed these restraints to produce molecular models of the potassium channel pore region, H5. The H5 sequence has been modeled either as a tetramer of membrane-spanning beta-hairpins, thus producing an eight-stranded beta-barrel, or as a tetramer of incompletely membrane-spanning alpha-helical hairpins, thus producing an eight-staved alpha-helix bundle. In total, restraints-directed modeling has produced 40 different configurations of the beta-barrel model, each configuration comprising an ensemble of 20 structures, and 24 different configurations of the alpha-helix bundle model, each comprising an ensemble of 24 structures. Thus, over 1300 model structures for H5 have been generated. Configurations have been ranked on the basis of their predicted pore properties and on the extent of their agreement with the biochemical data. This ranking is employed to identify particular configurations of H5 that may be explored further as models of the pore-lining region of the voltage-gated potassium channel pore.