The expression of functional ricin B-chain in Saccharomyces cerevisiae.

Yeast cells transformed with plasmids containing ricin B-chain coding sequences expressed this heterologous protein. When ricin B-chain was expressed in a form which resulted in its deposition in the yeast cytosol it formed insoluble aggregates which were devoid of galactose-binding activity. In contrast, when DNA fusions were constructed, in which the B-chain coding sequence was preceded by either the preproalpha-factor leader sequence or the native preproricin signal sequence, the recombinant B-chain products were soluble and biologically active. Both the homologous yeast signal peptide and the heterologous plant signal peptide directed the expressed product into the lumen of the yeast endoplasmic reticulum. As a result, the recombinant B-chain products were processed at the N-terminus, glycosylated and folded into an active conformation, presumably stabilized by correct intrachain disulphide bond formation.

Recombinant proricin binds galactose but does not depurinate 28 S ribosomal RNA.

Preproricin transcripts microinjected into Xenopus oocytes were expressed and the product was segregated by the oocyte endoplasmic reticulum and core glycosylated. Recombinant proricin was soluble, stabilised by intramolecular disulfide bonds and biologically active in that it could bind to immobilized lactose (selectin 2) or immobilized asialofetuin. Affinity-purified proricin did not catalyse the depurination of 28 S ribosomal RNA unless it was reduced, when slight but significant activity was observed. Gel filtration of the reduced proricin fraction showed that this depurination activity was not associated with proricin. The activity was apparently due to ricin A chain released by reduction from mature ricin which was, in turn, generated from proricin, presumably via endogenous oocyte endoprotease activity.

Elemental content of predigested liquid protein products.

Eight commercially available partially digested gelatin liquid products that have been used as a source of protein in dietary regimens for weight reduction and for protein supplementation were analyzed for their elemental composition. These products were prepared either by dry ashing or wet digestion techniques that were suitable for the analysis of individual elements by their respective analytical methods. Analytical methods used to assess the elemental content included inductively coupled argon plasma emission spectrometry for Ca, Mg, P, Fe, Zn, Cu, Mn, Ni, Mo, V, Be, Co, Cr, Tl, Al, Te, Sn and Sb; atomic absorption spectrophotometry for Na and K; hybride generation with atomic absorption spectrophotometry for Se and As; anodic stripping voltammetry for Cd and Pb; and ion selective electrode for F. The content of almost all of the elements in these products was extremely low compared with the amounts supplied daily for normal individuals by usual sources of dietary protein, the adult United States Recommended Daily Allowances, and typical intakes from the Total Diet Study. The following elements were not detectable in significant amounts in these products by inductively coupled argon plasma emission spectrometry: Mo, V, Be, Co, Cr, Tl, Al, Te, Sn, and Sb. The results suggest that use of these predigested liquid protein products as a sole source of nutriment will result in an inadequate intake of all of the essential elements. These products contained insignificant amounts of Ce and Pb, nonessential toxic elements.

Do children with a specific reading disability have a general serial-ordering deficit?

Two experiments are described in which reading disabled subjects and their normal controls were tested for their ability to abstract sequential regularities from a noisy background. Subjects were presented with an adaptation of the repeated digits task of Hebb (1961) and its spatial analogue the Corsi Blocks (Milner, B. Br. Med. Bull. 27, 272-277, 1971). Normal subjects had a digit span significantly better than their block span and also significantly better than that of the disabled readers. This suggested that normal subjects have better defined "storage filters" for digits or a specific advantage in the construction of 'transitory filters' for verbal material. There were no differences between groups on block span. On the repeated digit sequences normal readers abstracted a relatively stable "storage filter" in the first five recurrent trials whereas the disabled readers showed no discrimination until the last five recurrent trials. On the repeated blocks task the disabled group performed as well as normal readers. Taken together this date gives strong evidence for a specific deficit in verbal serial organization in disabled readers but does not support a general deficit in serial organization.

Transformation of plants via the shoot apex.

The terms "meristem" and "shoot tip" culture have often been indiscriminately interchanged. According to Cutter 1), the apical meristem refers to only that portion of the shoot apex lying distal to the youngest leaf primordium. The shoot apex, or shoot tip, consists of the apical meristem and one to three subjacent leaf primordia. True apical meristem culture of higher plants was first demonstrated by Smith and Murashige 2). The isolated tissues of the apical domes develop directly into plants, demonstrating the developmental automony of the angiosperm shoot apical meristem.

The planning of precision movements.

Two experiments are reported in which subjects were required to make rapid aiming movements to targets of various sizes. Probe reaction time (RT) procedures were used to investigate the preparation of the response to the target. It was proposed that if the precision of movement was planned in advance, this would be reflected by the lengthening of RTs to probes presented during the latency phase of the response. The more precise the movement (to smaller targets) the longer will be the delays to the probes. The results generally supported the prediction and the probe RTs were correlated with target size. There was also some evidence that the probe was lengthening during the movement and in the region of the target.

Lead, fluoride, and other elements in bonemeal supplements.

The Pb, Cd, F, Al, Cr, Cu, Fe, Mn, Mo, Ni, Ti, and Zn content of 20 commercial bonemeal supplements was determined. Samples were mineralized with nitric and perchloric acids prior to determination of all elements except F, for which a diffusion method was used. Pb and Cd were determined by differential pulse anodic stripping voltammetry, F was measured using an ion selective electrode, and all other elements were determined by inductively coupled argon plasma spectroscopy. The mean recoveries of Pb and F were 97 and 99%, respectively. The concentration range of Pb was 1.5-8.7 microgram/g. Cd was quantitated in only one sample at a level of 2.5 microgram/g; all other samples were estimated to contain less than 0.05 microgram Cd/g. The concentration of F ranged from 261 to 921 microgram/g.

Death in practice.

For a nationwide study of terminal care, Gallup Polls took a sample of 950 general practitioners. Fourteen questionnaires could not be delivered; 376 doctors (40 per cent of the register) returned forms; 313 doctors (33.4 per cent) provided information on 301 home deaths and 292 hospital deaths and responded also to statements about care of the dying.The patients who died at home were well supported by the general practitioner and the family and neighbourhood network. Control of pain was perceived to be better at home. Patients dying at home were more likely to be aware of their impending death. General practitioners usually discussed the imminence of death with relatives, but few relatives and patients raised the question of terminating life. All the available major services were under-used. There was support for more hospices and for more spending on social services. Postgraduate medical education on care of the dying was considered to be inadequate.

Finger injuries involving stationary exercise bicycles.

Injuries to children related to exercise bicycles can result in significant morbidity, but are almost entirely preventable. To forestall these injuries, manufacturers should continue to focus their attention on improved design and developing safer equipment. In addition, pediatricians and other primary care providers need to play a role in educating parents and the community about the possible hazards that exercise bicycles present to young children.

Inducible cell ablation in Drosophila by cold-sensitive ricin A chain.

We have developed a system for temperature-inducible killing of specific cells in the fruitfly Drosophila melanogaster. The system overcomes many of the limitations of existing cell ablation methods and is in principle applicable to any non-homeothermic eukaryote. Temperature-sensitive and cold-sensitive mutations in the ricin toxin A chain (RTA) of castor bean were generated in yeast. One cold-sensitive mutation, RAcs2, produced temperature-dependent ablation of eye cells in Drosophila when expressed under control of the eye-specific sev enhancer. At 29 degrees C, cell death was observed within 7 hours in the developing eye and no obvious toxic effects were observed elsewhere; at 18 degrees C, extremely low toxicity was observed. DNA sequencing of RAcs2 revealed a single amino acid substitution in the RTA active site cleft.

Hot leaching of ceramic and enameled cookware: collaborative study.

A hot leach method published by the World Health Organization for determining Pb and Cd in ceramic and enameled ware was collaboratively studied in 14 laboratories. The method consisted of heating a solution of 4% acetic acid at the boil for 2 h in 6 samples of specially glazed ceramic ware and 6 samples of special enameled ware. The acid was allowed to cool and stand in contact with the ware for an additional 22 h. At the end of the 2 h heating period and again at the end of the 24 h period, the leach solution was assayed by atomic absorption spectrophotometry for Pb and Cd. Ruggedness testing before the collaborative study revealed that: (1) the quantity of metal released increased as the average leach temperature approached the boiling point, (2) the quantity of Pb and Cd released from enameled ware increased linearly with time during 7 h of boiling, (3) the concentration of acid could be varied from 2 to 6% with no apparent effect on the amount of metal released, and (4) the room temperature (RT) contact period lasting from 22 to 120 h (after the 2 h heating period) did not increase the amount of metal released. The quantities of Pb and Cd measured by the collaborators at the end of 2 h of heating and after 24 h of total contact were essentially identical. The amounts of Pb and Cd were not related to the quantity of acid solution required to restore the leach solution volume to its initial value after boiling. The collaborative results showed that the period of standing at RT is not necessary and probably can be eliminated from the method. The modified method, which requires analysis of the leach solution immediately after the 2 h heating period, has been adopted interim official first action by AOAC.

Opines stimulate induction of the vir genes of the Agrobacterium tumefaciens Ti plasmid.

Upon incubation of Agrobacterium tumefaciens A348 with acetosyringone, the vir genes encoded by the Ti (tumor-inducing) plasmid are induced. The addition of certain opines, including octopine, nopaline, leucinopine, and succinamopine, enhanced this induction 2- to 10-fold. The compounds mannopine, acetopine, arginine, pyruvate, and leucine did not stimulate the induction of the vir genes to such an extent. The enhancement of vir gene induction by opines depended on acetosyringone and the genes virA and virG. Opines stimulated the activity of the vir genes, the double-stranded cleavage of the T (transferred)-DNA at the border repeat sequences, and the production of T-strands by the bacterium. The transformation efficiency of cotton shoot tips was markedly increased by the addition of acetosyringone and nopaline at the time of infection.

Influence of automatic dishwashings and scrubbings on release of lead from glazed ceramicware.

Use of lead glazes on ceramicware and the release of lead from finished glazes are reviewed. Single units of ceramicware with initial lead leach levels from less than 0.1 to 470 micrograms/mL were subjected to multiple automatic dishwashings with intermediate scrubbing of the ware by plastic fiber pads. Ware was periodically leached with 4% acetic acid solution at room temperature for 24 h, and the solutions were analyzed for lead. Release of lead under these conditions is discussed. Results indicated that neither passive leachings nor multiple dishwashings and scrubbings can predict future lead-release characteristics of glazes and decorations.

Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes.

Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified. We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.