Nonreciprocal Pseudotyping: Murine Leukemia Virus Proteins Cannot Efficiently Package Spleen Necrosis Virus-Based Vector RNA

It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Ψ) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Ψ, but MLV can recognize only MLV Ψ. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.     

ASSESSING THE LIMITS OF RANDOM AMPLIFIED POLYMORPHIC DNAs (RAPDs) IN SEAWEED BIOGEOGRAPHY1

As judged by comparison with other molecular data sets, random amplified polymorphic DNA (RAPD) data are robust in identifying large-scale biogeographic populations that range from hundreds to thousands of kilometers apart. As the geographical scale is shifted downward, however, RAPD data often fail. This is because RAPD data are inherently “noisy” as a result of technical artifacts and reproducibility problems associated with non-independence of bands, “missing” bands, and the presence of de novo bands, all of which contribute to scoring errors in the data set. To estimate the contribution of these error factors in algal phylogeographic studies, segregation of RAPD bands in tetrasphorophytic and gametophytic parents, their natural and synthetic offspring, and self-cycled tetrasporophytes were compared in Lophocladia trichoclados (Mertens in C. Agardh) Schmitz and to a limited extent in Digenea simplex (Wulfen) C. Agardh. Wide-ranging biogeographic populations of D. simplex were compared as were mixed populations of tetrasporophytes and gametophytes. Results show that nested priming can lead to some nonindependence of bands but that this probably does not significantly contribute to scoring error. Southern analysis using individual RAPD bands as probes revealed that up to 16% of visually nondetectable bands are actually present but that the random distribution of the error contributes uniformly across the data set. Non-parental (de novo in offspring) and parental (not present in offspring) bands may contribute substantially to the scoring error in tetrasporophytes, gametophytes, and self-cycled tetrasporophytes. The presence of tetrasporophytes and gametophytes in a sample is not important in large-scale phylogeographic studies but does affect within-clade variation at smaller scales. We conclude that the overall level of error remains roughly constant at probably between 5 and 10%, which is not a problem at large biogeographic scales where the phylogenetic signal is strong. Finally, some unexpectedly large abberations in RAPD banding patterns among life stages in L. trichoclados were observed that cannot be explained by methodological artifacts alone due to comparisons with synthetic offspring controls. The possibility that carpospore amplification may not always involve a simple mitotic process is discussed.     

Appearance of contractile activity in muscular dysgenesis (mdgmdg) mouse myotubes during coculture with normal spinal cord cells

Muscular dysgenesis (mdg) in the mouse is an autosomal recessive mutation expressed in the homozygous mutant as lack of skeletal muscle contraction. To test the ability of normal neurons to form neuromuscular contacts with, and/or possibly induce contractions in $\text{mdg}\text{mdg}$ muscle, dispersed cell cultures of normal and dysgenic muscle from newborn mice were cocultured with normal embryonic rat, mouse, and chick dissociated spinal cord cells. Contraction was induced in $\text{mdg}\text{mdg}$ muscle 1 to 10 days (depending upon the species of the neuronal source) following establishment of the cocultures. Control experiments indicated that the dispersed spinal cord preparations were free of myoblasts capable of fusing with $\text{mdg}\text{mdg}$ muscle. The establishment of neuromuscular contacts in the rat neuron cocultures was monitored by cytochemical staining of acetylcholinesterase (AChE), autoradiography of ¹²⁵I-α-bungarotoxin-bound acetylcholine receptors (AChR), and electrophysiological study of muscle membrane activity. Patches of high AChE activity were similar in size and distribution to high-density clusters of AChR on both control and $\text{mdg}\text{mdg}$ myotubes cocultured with rat neurons. The resting membrane potentials of normal myotubes and those of $\text{mdg}\text{mdg}$ myotubes in the presence of neurons were similar (? ?52 mV). The mepp frequency and the mepp amplitude distribution were the same for both control and mutant cocultured muscle. Thus, normal rat spinal cord neurons were capable of forming normal, functional neuromuscular junctions with $\text{mdg}\text{mdg}$ myotubes, and contractions were induced under coculture conditions, in otherwise noncontracting mutant muscle.     

Expected and observed proportion of subjects excluded from paternity by blood phenotypes of a child and its mother in a sample of 171 families

The proportion of exclusion for a given mother-child pair is the proportion of males excluded from the paternity of this child of a known mother and may be calculated given both the child's and mother's phenotypes and the population gene frequencies. Its expected value in the population is equal to the probability of exclusion, which expresses a laboratory's capability to exclude from paternity nonbiological fathers.

In a sample of 171 families examined for 20 genetic systems at the National Blood Group Reference Laboratory, 25 exclusions of putative fathers were detected. The ranking by efficiency of the systems used in these exclusions fits the “expectation of their efficiency,” and the average proportion of males excluded by the child's and mother's phenotypes is not different from the expected proportion. Additionally, the repetition of exclusions in an incompatible putative father-mother-child trio is not dependent on the overall proportion of males excluded by the mother and the child, but rather on some high values of the proportion of excluded men in some specific systems.

Here, formulas and some factors modifying these parameters as well as a more efficient sequence of examinations to exclude paternity than has previously been used are given. Using this sequence, laboratories which carry out several analyses per day can work by levels of five examinations at a time, done in a particular order, to obtain a rather rapid exclusion of certain families.

     

Human transferrin (Tf) and group-specific component (Gc) subtypes in Tunisia

Summary Simultaneous subtyping of two genetic markers—group-specific component (Gc) and transferrin (Tf)—by electrofocusing enabled us to compute the following gene frequencies for the Tunisian population: Gc ^IS .0.525; Gc ^IF , 0.260; Gc ², 0.215; Tf ^CI , 0.770; Tf ^C2 , 0.215; Tf ^D1 , 0.015.The frequencies of Tf ^D , Tf ^C2 , and Gc ¹ are higher than those found in Caucasoid populations and can be explained by Negroid contribution. A selective advantage related to the metabolic role of this vitamin D-binding protein does not seem very likely for any particular Gc type or subtype. It is postulated that the differences in the frequencies of the Gc alleles might be related to selective advantage for genes belonging to other genetic systems originally closely linked to either Gc ¹ or to Gc ² alleles.This work was supported in part by the Faculté de Pharmacie et de Médecine Dentaire of Monastir and by a grant from the Ambassade de France in Tunisia     

Some ring-opening reactions of a diepoxide derived from (−)-quinic acid

A useful preparative route to nitrogen-containing, carbocyclic derivatives is described from (−)-quinic acid. (−)-Quinic acid was converted via the 3,4-O-cyclohexylidene-lactone into 1- -3-O-tosyl-5-C-tosyloxymethylcyclohexane-1,2,5/3-tetrol (5) by sequential reduction with sodium borohydride, toluene-p-sulphonylation, and acid hydrolysis. Reaction of the disulphonate 5 with methanolic sodium methoxide afforded 1- -1,2:5,7-dianhydro-5-C-hydroxymethylcyclohexane-1,2,3,5/0-tetrol (6). The ring-opening reactions of the diepoxide 6 with azide ion furnished a mixture of two diazides 9 and 13 in the ratio 4 to 1. The structure and conformation of the derived dibenzoates 10 and 14 have been determined by n.m.r. spectroscopy.     

WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27

The recently discovered cytokine IL-27 belongs to the IL-6/IL-12 family of cytokines and induced proliferation of naive CD4(+) T cells and the generation of a Th1-type adaptive immune response. Although binding of IL-27 to the cytokine receptor WSX-1 was demonstrated, this interaction proved insufficient to mediate cellular effects. Hence, IL-27 was believed to form a heteromeric signaling receptor complex with WSX-1 and another, yet to be identified, cytokine receptor subunit. In this study, we describe that WSX-1 together with gp130 constitutes a functional signal-transducing receptor for IL-27. We show that neither of the two subunits itself is sufficient to mediate IL-27-induced signal transduction, but that the combination of both is required for this event. Expression analysis of WSX-1 and gp130 by quantitative PCR suggests that IL-27 might have a variety of cellular targets besides naive CD4(+) T cells: we demonstrate gene induction of a subset of inflammatory cytokines in primary human mast cells and monocytes in response to IL-27 stimulation. Thus, IL-27 not only contributes to the development of an adaptive immune response through its action on CD4(+) T cells, it also directly acts on cells of the innate immune system.     

A sugar ester and an iridoid glycoside from Scrophularia ningpoensis

From cytotoxic extracts of the roots of Scrophularia ningpoensis Hemsl. (Scrophulariaceae) a new sugar ester, ningposide D (3-O-acetyl-2-O-p-methoxycinnamoyl-alpha(beta)-L-rhamnopyranose) (1) and a new iridoid glycoside, scrophuloside B4 (6-O-(2'-O-acetyl-3'-O-cinnamoyl-4'-O-p-methoxycinnamoyl-alpha-L-rhamnopyranosyl) catalpol) (2) along with known compounds: oleanonic acid (3), ursolonic acid (4), cinnamic acid (5), 3-hydroxy-4-methoxy benzoic acid (6), 5-(hydroxymethyl)-2-furfural (7) and beta-sitosterol (8) were isolated. The structures of the new compounds were elucidated by spectral data (1, 2D NMR, EI, HRESI-MS and MS/MS). Oleanonic acid (3) and ursolonic acid (4) were found to be cytotoxic against a series of human cancer cell lines with IC50=4.6, 15.5 microM on MCF7; 4.2, 14.5 microM on K562; 14.8, 44.4 microM on Bowes; 24.9, 43.6 microM on T24S; 61.3, 151.5 microM on A549, respectively. Beta-sitosterol (8) inhibited Bowes cells growth at IC50=36.5 microM. Scrophuloside B4 (2) showed activity on K562 and Bowes cells at IC50=44.6, 90.2 microM, respectively.     

Phosphorylation control of the ubiquitin ligase Cbl is conserved in choanoflagellates

Cbl proteins are E3 ubiquitin ligases specialized for the regulation of tyrosine kinases by ubiquitylation. Human Cbl proteins are activated by tyrosine phosphorylation, thus setting up a feedback loop whereby the activation of tyrosine kinases triggers their own degradation. Cbl proteins are targeted to their substrates by a phosphotyrosine‐binding SH2 domain. Choanoflagellates, unicellular eukaryotes that are closely related to metazoans, also contain Cbl. The tyrosine kinase complement of choanoflagellates is distinct from that of metazoans, and it is unclear if choanoflagellate Cbl is regulated similarly to metazoan Cbl. Here, we performed structure‐function studies on Cbl from the choanoflagellate species Salpingoeca rosetta and found that it undergoes phosphorylation‐dependent activation. We show that S. rosetta Cbl can be phosphorylated by S. rosetta Src kinase, and that it can ubiquitylate S. rosetta Src. We also compared the substrate selectivity of human and S. rosetta Cbl by measuring ubiquitylation of Src constructs in which Cbl‐recruitment sites are placed in different contexts with respect to the kinase domain. Our results indicate that for both human and S. rosetta Cbl, ubiquitylation depends on proximity and accessibility, rather than being targeted toward specific lysine residues. Our results point to an ancient interplay between phosphotyrosine and ubiquitin signaling in the metazoan lineage.     

Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue

The development of a barley ( Hordeurn vulgare L.) transformation system made it possible to consider the use of maize Activator/Dissociation ( Ac/Ds ) transposable elements for gene tagging in transgenic barley plants. However, barley transformation is time-consuming, and therefore a simple transient assay for Ac/Ds activity in intact barley tissues was developed to test the components of a proposed gene tagging system, prior to their stable introduction into plants. In this assay, barley scutellar tissue is co-transformed with constructs containing the maize Ac transposase gene and an Escherichia coli uid A reporter gene ( Gus ), the expression of which is interrupted by a maize Ds element. In transformed barley scutellar cells, Ac transposase-mediated excision of the Ds element generates a functional Gus gene, leading to histochemically detectable GUS activity. Characterization of the excision products showed that they had a pattern of nucleotide deletions and/or transversions similar to that found in maize and other heterologous plant systems. In addition, although contrary to the situation observed in heterologous dicot systems, efficient Ds excision in barley, a heterologous monocot system, appears to be inversely associated with Ac copy number, a finding similar to the Ac dosage effects observed in maize. The transient assay was used to demonstrate functional transposase activity in barley callus lines stably transformed with an Ac transposase gene.