Green fluorescent antibodies: novel in vitro tools

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.     

Shared salinity tolerance invalidates a test for the malaria vector Anopheles farauti s.s. on Guadalcanal, Solomon Islands

Among the Punctulatus Group of Anopheles mosquitoes (Diptera: Culicidae), first-instar larvae of the medically unimportant freshwater Anopheles farauti species No. 7 survives a seawater tolerance test (SST) that was previously thought to be diagnostic for the saltwater-tolerant malaria vector species, An. farauti Laveran s.s. Salt tolerance in these two closely related isomorphic species appears to be a shared derived character within the Farauti Complex. Failure to differentiate An. farauti s.s. from An. farauti No. 7 will overestimate potential malaria vector numbers and waste limited larval control resources. Use of the SST should therefore be discontinued on Guadalcanal and other techniques such as allozyme electrophoresis used instead.     

Reconciling different models of forebrain induction and patterning: a dual role for the hypoblast

Several models have been proposed for the generation of the rostral nervous system. Among them, Nieuwkoop's activation/transformation hypothesis and Spemann's idea of separate head and trunk/tail organizers have been particularly favoured recently. In the mouse, the finding that the visceral endoderm (VE) is required for forebrain development has been interpreted as support for the latter model. Here we argue that the chick hypoblast is equivalent to the mouse VE, based on fate, expression of molecular markers and characteristic anterior movements around the time of gastrulation. We show that the hypoblast does not fit the criteria for a head organizer because it does not induce neural tissue from na?ve epiblast, nor can it change the regional identity of neural tissue. However, the hypoblast does induce transient expression of the early markers Sox3 and Otx2. The spreading of the hypoblast also directs cell movements in the adjacent epiblast, such that the prospective forebrain is kept at a distance from the organizer at the tip of the primitive streak. We propose that this movement is important to protect the forebrain from the caudalizing influence of the organizer. This dual role of the hypoblast is more consistent with the Nieuwkoop model than with the notion of separate organizers, and accommodates the available data from mouse and other vertebrates.     

Regulation of Tiam1 nucleotide exchange activity by pleckstrin domain binding ligands

Rho family GTPases play roles in cytoskeletal organization and cellular transformation. Tiam1 is a member of the Dbl family of guanine nucleotide exchange factors that activate Rho family GTPases. These exchange factors have in common a catalytic Dbl homology and adjacent pleckstrin homology domain. Previous structural studies suggest that the pleckstrin domain, a putative phosphoinositide-binding site, may serve a regulatory function. We identified ascorbyl stearate as a compound that binds to the pleckstrin domain of p120 Ras GTPase-activating protein. Furthermore, ascorbyl stearate appears to be a general pleckstrin domain ligand, perhaps by mimicking an endogenous amphiphilic ligand. Tiam1 nucleotide exchange activity was greatly stimulated by ascorbyl stearate. Certain phosphoinositides also stimulated Tiam1 activity but were less potent than ascorbyl stearate. Tiam1 contains an additional N-terminal pleckstrin domain, but only the C-terminal pleckstrin domain was required for activation. Our results suggest that the pleckstrin domains of Dbl-type proteins may not only be involved in subcellular localization but may also directly regulate the nucleotide exchange activity of an associated Dbl homology domain. In addition, this paper introduces ascorbyl stearate as a pleckstrin domain ligand that can modulate the activity of certain pleckstrin domain-containing proteins.     

A synthetic peptide ligand of neural cell adhesion molecule (NCAM) IgI domain prevents NCAM internalization and disrupts passive avoidance learning

The neural cell adhesion molecule (NCAM) mediates cell adhesion and signal transduction through trans-homophilic- and/or cis-heterophilic-binding mechanisms. Intraventricular infusions of anti-NCAM have revealed a functional requirement of NCAM for the consolidation of memory in rats and chicks in a specific interval 6-8 h after training. We have now extended these studies to a synthetic peptide ligand of NCAM (C3) with an affinity for the IgI domain and the capability of inhibiting NCAM-mediated neurite outgrowth in vitro. Intraventricular administration of a single 5 microg bolus of C3 strongly inhibited recall of a passive avoidance response in adult rats, when given during training or in the 6-8-h posttraining period. The effect of C3 on memory consolidation was similar to that obtained with anti-NCAM as the amnesia was not observed until the 48-h recall time. The unique amnesic action of C3 during training could be related to disrupted NCAM internalization following training. In the 3-4-h posttraining period NCAM 180, the synapse-associated isoform, was down-regulated in the hippocampal dentate gyrus. This effect was mediated by ubiquitination and was prevented by C3 administration during training. These findings indicate NCAM to be involved in both the acquisition and consolidation of a passive avoidance response in the rat. Moreover, the study provides the first in vivo evidence for NCAM internalization in learning and identifies a synthetic NCAM ligand capable of modulating memory processes in vivo.     

Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Grip strength changes over 27yr in Japanese-American men

The aim of thisstudy was to describe changes in grip strength over a follow-up periodof ~27 yr and to study the associations of rate of strength declinewith weight change and chronic conditions. The data are from theHonolulu Heart Program, a prospective population-based studyestablished in 1965. Participants at exam1 were 8,006 men (ages 45-68 yr) who were ofJapanese ancestry and living in Hawaii. At follow-up, 3,741 men (agerange, 71-96 yr) participated. Those who died before the follow-upshowed significantly lower grip-strength values at baseline than didthe survivors. The average annualized strength change among thesurvivors was 1.0%. Steeper decline (>1.5%/yr) wasassociated with older age at baseline, greater weight decrease, andchronic conditions such as stroke, diabetes, arthritis, coronary heartdisease, and chronic obstructive pulmonary disease. The risk factorsfor having very low hand-grip strength at follow-up, here termedgrip-strength disability (21 kg, the lowest 10th percentile), werelargely same as those for steep strength decline. However, theage-adjusted correlation between baseline and follow-up strength wasstrong (r = 0.557, P < 0.001); i.e., those who showedgreater grip strength at baseline were also likely to do so 27 yrlater. Consequently, those in the lowest grip-strength tertile atbaseline had about eight times greater risk of grip-strength disabilitythan those in the highest tertile because of their lower reserve ofstrength. In old age, maintenance of optimal body mass may help preventsteep strength decrease and poor absolute strength.

     

Ethnic Identity and Power: Cultural Contexts of Political Action in School and Society.

Ethnic Identity and Power. Cultural Contexts of Political Action in School and Society. Yali Zou and Enrique T. Trueba. eds. Albany: State University of New York Press, 1998. 452 pp.