Neuronal binding of tetanus toxin compared to its ganglioside binding fragment (H(c))

The non-toxin 50 kD C-terminus peptide of the heavy chain of tetanus H(c) contains the ganglioside binding domain of tetanus toxin (TTX). H(c) retains much of the capacity of tetanus toxin for binding internalization and transport by neurons. For this reason tetanus H(c) has been studied as a vector for delivery of therapeutic proteins to neurons. We directly compared H(c) and TTX in the capacity to bind and be internalized by neurons by ELISA. Primary cultures of dissociated fetal cortical neurons were incubated with equimolar amounts of TTX or H(c). Neuronal associated tetanus protein was 4-8 fold greater on a molar basis with tetanus toxin compared to H(c) (1 h incubation). This increase in neuronal tetanus protein was evident with incubation in concentrations from 0.1 microM to 2 microM. There were greater amounts of TTX delivered to the cultured cells at both 0 degrees C (representing membrane bound tetanus protein) and 37 degrees C (bound and internalized tetanus protein). Unlike H(c), TTX showed significant continued accumulation of protein with increasing incubation durations. Neuronal associated TTX increased 2-3 fold over incubation times ranging from 1 to 8 h. Tetanus toxin appears to be clearly superior to the ganglioside binding fragment (H(c)) in the capacity for neuronal binding and internalization. Atoxic tetanus proteins containing additional molecular domains as well as H(c) may be more suitable vectors for linkage with therapeutic proteins and delivery to neurons.     

Identification of a family of human F-box proteins.

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2] [3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2] [4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins.     

A chloroplast DNA Phylogenetic study of the eastern Asia-eastern North America disjunct section Rytidospermum of Magnolia (Magnoliaceae)

Chloroplast DNA restriction site variation of ten endonucleases was examined among all ten species or varieties of the eastern Asia–eastern North America disjunct section Rytidospermum of Magnolia. Representatives from seven of the other ten sections of Magnolia and four related genera (Liriodendron, Manglietia, Michelia, and Talauma) were also included in the survey. A cladistic analysis of 200 variable sites using Wagner parsimony yielded 11 equally most parsimonious trees with a consistency index of 0.793 and a retention index of 0.870. The section Rytidospermum is polyphyletic in these trees. Magnolia tripetala from the southeastern U.S. is the only American species that has a sister relationship to the Asian taxa, M. hypoleuca, M. officinalis var. officinalis, M. officinalis var. biloba, and M. rostrata. Other American taxa in the section fall into two lineages, with M. macrophylla var. macrophylla, M. macrophylla var. ashei, and M. macrophylla var. dealbata in one, and M. fraseri var. fraseri and M. fraseri var. pyramidata in the other. They are not related to the Asian species as previously believed. The relationships revealed here are in agreement with morphological, allozymic, and cross compatibility data. These results demonstrate that a robust phylogenetic hypothesis is an important prerequisite for understanding biogeographic patterns.     

A rapid and sensitive micro-assay for the enzymatic determination of plasma and lipoprotein cholesterol

A rapid and inexpensive micro-assay for determining cholesterol in plasma and isolated lipoprotein fractions has been established which utilizes a commercially available enzymatic reagent with semi-automated instruments and microtiter plates. The assay is sensitive, precise, and easy to perform. The color development is linear from 0.4 to 20 micrograms cholesterol/well, with sample volumes of 2 to 100 microliters. Inter- and intra-assay variability yielded coefficients of variation (CV) of 2.75% (n = 51) and 1.09% (n = 32), respectively. The concentrations of total plasma and lipoprotein cholesterol (d greater than 1.006 g/ml) obtained with this method were compared with those analyzed in a lipid laboratory standardized to the Centers for Disease Control. The correlation coefficients between the two methods were 0.976 and 0.964, respectively. For total high density lipoprotein (HDL) and the HDL3 subfraction, inter-assay variability was 4.12% and 6.33% (n = 27), respectively; the intra-assay variability was 2.79% and 4.19% (n = 12).     

Analysis of metal incorporation during overexpression of Clostridium pasteurianum rubredoxin by electrospray FTICR mass spectrometry

The rate of production of Clostridium pasteurianum rubredoxin overexpressed in Escherichia coli was examined by electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Previous work had shown that this heterologous expression resulted in isolation of both iron-containing (FeRd) and zinc-containing (ZnRd) rubredoxins. In the present work, minimally processed cell lysates of E. coli were analyzed in order to monitor the production of FeRd and ZnRd. The sensitivity of the measurement favored FeRd relative to ZnRd, and this differential sensitivity was quantitated using previously separated and purified rubredoxins. A time course study indicated that ZnRd and FeRd are produced simultaneously during overexpression, but at different rates. The ratio of the concentration of ZnRd to FeRd increased in a linear fashion during 3 h following induction of overexpression. Since only FeRds have been reported from native bacteria and archaea, the data suggest that either Zn2+ is sequestered from rubredoxins during native biosynthesis or that ZnRds may have escaped detection in the native microorganisms. ESI-FTICR mass spectrometry is shown to be a useful tool for monitoring metal insertion during protein biosynthesis.