Ricin B chain fragments expressed inEscherichia coli are able to bind free galactose in contrast to the full length polypeptide

Deleted forms of ricin B chain (RTB) containing only one of the two galactose binding sites were produced inE. coli and targeted to the periplasm by fusion to theompA orompF signal sequences. The proteins were then isolated from the periplasm and their sugar binding properties assessed. Previous studies investigating the properties of such proteins produced inXenopus laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugars, unlike the full-length unglycosylated proteins. When produced inE. coli however we found that only one, EB733, of a number of deleted forms of RTB closely related to those previously produced inXenopus laevis oocytes, bound to simple sugars. All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which has been previously utilized to measure binding of lectins to the terminal galactose residues of glycoprotein oligosaccharides. However, in contrast to glycosylated RTB, binding of the deletion mutants could be competed to only a small degree or not at all with galactose. The only deletion mutant observed to bind to free galactose when produced inE. coli corresponded closely to the complete domain 2 of RTB. It is assumed that this mutant forms a stable structure similar to that of the C-terminal domain in the full-length protein. The structural integrity of EB733 was not only suggested by its sugar binding properties and solubility but also by its consistently higher level of expression and the absence of any apparent susceptibility toE. coli proteases.Abbreviations RTA ricin toxin A chain - RTB ricin toxin B chain - ER endoplasmic reticulum - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - IPTG isopropyl -d-thiogalactopyranoside     

Predation on primates: Ecological patterns and evolutionary consequences

It has long been thought that predation has had important ecological and evolutionary effects on primates as prey. Predation has been theorized to have been a major selective force in the evolution of hominids.¹ In modern primates, behaviors such as active defense, concealment, vigilance, flight, and alarm calls have been attributed to the selective pressures of predation, as has group living itself. It is clear that primates, like other animals, have evolved ways to minimize their risk of predation. However, the extent to which they have been able to do so, given other constraints of living such as their own need to acquire food, has not yet been resolved. Perhaps most hotly debated is whether predation has been the primary selective force favoring the evolution of group living in primates. Part of the difficulty in resolving the debate lies in a paucity of direct evidence of predation. This is regrettable yet understandable since primatologists, by definition, focus on the study of primates, not predators of primates (unless these are also primates). Systematic direct evidence of the effects of predation can best be obtained by studying predators that are as habituated to observers as are their primate prey. Until this is done, we must continue to rely on opportunistic accounts of predation and predation attempts, and on systematically obtained indirect evidence. Such data reveal several interesting patterns: (1) although smaller primates may have greater predation rates than larger primates, even the largest primates are not invulnerable to predation; (2) the use by primates of unfamiliar areas can result in higher predation rates, which might be one pressure favoring philopatry, or site fidelity; (3) arboreal primates are at greater risk of predation when they are more exposed (at forest edges and tops of canopies) than in more concealed locations; (4) predation by mammalian carnivores may often be episodic; and (5) terrestrial primates may not experience greater predation than arboreal primates.     

Function of the upstream hypersensitive sites of the chicken beta-globin gene cluster in mice.

We have shown previously that the chicken beta A-globin gene, with its 3' enhancer, is expressed in a copy number-dependent manner in transgenic mice. The expression level was low but increased approximately 6-fold upon inclusion of 11 kb of upstream DNA containing four DNase I hypersensitive sites. To study the effect of the individual upstream hypersensitive sites on transgene expression, we produced lines of mice in which the individual upstream sites were linked to the beta A gene and enhancer. RNA levels were measured in blood from adult animals. With each of these four constructs, the level of transgene RNA per DNA copy varied over a > 20-fold range. These data suggest that addition of a hypersensitive site to the beta A-globin/enhancer region abrogates its position independent expression. The average beta A-globin expression per copy in the lines carrying an upstream site was comparable with that in lines without an upstream site. Thus, no single upstream hypersensitive site accounts for the higher level of beta A-globin expression seen in mice containing the complete upstream region. We had shown previously that control of the chicken beta-globin cluster is distributed between at least two regions, the beta A/epsilon enhancer and the upstream region. Our current results suggest that the control mediated by the upstream DNA is itself distributed and is not due to a single hypersensitive site.     

Behavioral and neurochemical changes in the dopaminergic system after repeated cocaine administration

In order to determine whether repeated cocaine administration produced persistent changes in dopamine (DA) receptor binding and release consistent with behavioral sensitization, rats were treated with either cocaine (25 mg/kg ip) or saline twice daily for 14 consecutive days followed by a 3-d withdrawal period. The DA transporter site was assayed using [³H]GBR 12935, whereas D₁ and D₂ sites were assayed using [³H]SCH 23390 and [³H]spiperone, respectively. The density (B _max) of the DA transporter binding sites in the ST of the cocaine-treated group increased significantly (p<0.05) over controls 3 d after the last injection, whereas the density of striatal D₁ and D₂ binding sites remained unchanged. The DA transporter in the nucleus accumbens (NA) was also studied with [³H]GBR 12935 and was unchanged following drug treatment. D₁ and D₂ binding parameters for the NA were not determined in this study. Furthermore, cocaine administration did not affect the affinities (K _d ) of the radioligands used to label the transporter, D₁, or D₂ sites in any of the studies performed. In addition, striatal DA release was measured using in vivo microdialysis in anesthetized rats. Linear regression analysis on maximal decreases in DA release after apomorphine (0.02, 0.2, and 2.0 mg/kg sc) injection showed no difference in the functional capacity of the ST to modulate DA transmission between control and treated groups. Moreover, animals pretreated with cocaine showed a significant (p<0.01) decrease in locomotor activity (LA) after a presynaptic, autoregulating dose of apomorphine (0.03 mg/kg sc) was given. These results suggest that the effects seen after repeated exposure to cocaine may be regulated, in part, by changes in striatal DA transporter binding site densities and not necessarily by DA-releasing mechanisms or D₁ and D₂ receptor modification.     

Heat-inducible expression of FLP gene in maize cells

The soybean heat-shock gene promoter ( Gmhsp 17.5-E ) has been used to direct expression of gusA and FLP genes in maize cells. At inducible temperatures, in transient expression assays, gusA gene expression controlled by the heat-shock promoter is about 10-fold higher than the expression directed by the CaMV 35S promoter. The Gmhsp 17.5-E promoter preserves its regulatory functions in heterologous maize cells after random integration into genomic DNA.
Heat-shock inducible expression of the FLP gene was investigated by co-transformation of the FLP expression vector (pHsFLP) and a recombination test vector (pUFNeo-FmG) into maize protoplasts. Co-transformed protoplasts were incubated at 42°C for 2 h. This treatment induced recombination of 20–25% of the available FRT sites in transient assays. As a result of heat-shock treatment of stably co-transformed maize cells, activation of gusA gene expression and an associated decrease or elimination of NPT-II activity in transgenic maize lines was observed. Molecular evidence was obtained of the expected DNA excision process catalyzed by the FLP protein in maize transgenic cells. Thus, the experiments presented in this paper indicate that the FLP protein can recognize and subsequently recombine the FRT target sites that had integrated into plant genomic DNA, and that regulated expression of the FLP gene is possible in maize cells using the soybean heat-shock promoter.     

Diet and Health

The role of diet in personal health maintenance is important whether a person is trying to stay healthy or to treat diet-related diseases such as hypercholesterolemia, hypertension, diabetes mellitus or obesity. Dietary recommendations include limiting fat to 30% and protein to 20% of total calories, with the remaining 50% coming from carbohydrate. Maintaining dietary changes for long periods is very difficult for many persons. Specific self-management skills may ease the task.     

The ultrastructure of campaniform sensilla on the eye of the cricket,Gryllus campestris

Summary The structure of the campaniform sensilla of the cricket eye was investigated by light and electron microscopy. Each sensillum is innervated by a single bipolar neuron. Its axon extends through the retina into a side-branch of the nervus tegumentarius. The dendrite extends through a cuticular channel to the surface of the cornea. The distal part of the dendrite, the sensory process, contains a tubular body and is attached to a cuticular cap which is obliquely inserted into the exocuticle between the corneal lenslets. Some particular structural features as well as the function of the campaniform sensillum of the cricket eye are discussed.Supported by the Deutsche Forschungsgemeinschaft, grant Ho 463/10The authors are indebted to Prof. H. Altner, University of Regensburg, and Mrs. Evelyn Thury, Contron GmbH, München for use of the scanning electron microscope facilities     

Antibodies distinguishing between intact and alkali-hydrolyzed 7-methylguanosine

Antibodies specific for intact 7-methylguanosine (m7G) were induced in rabbits and mice by immunization with nucleoside-BSA or nucleoside-hemocyanin conjugates. Since m7G undergoes alkali-catalyzed hydrolytic fission of the purine ring, modifications were made in the procedure for conjugation of m7G to proteins. After periodate oxidation, m7G was incubated with protein at pH 9.1 at 4 degrees C for one hour during which the nucleoside was found to be stable. Reduction of the Schiff base was done with t-butylamine borane for 30 minutes, and the conjugated protein was isolated quickly by gel filtration at pH 7.2. Both rabbits and mice produced antibodies that readily distinguished between the intact and hydrolyzed m7G. Antibody specificity depended largely on the presence of an intact 7-substituted imidazole ring and some cross-reaction occurred with 7-methylinosine. A weaker reaction occurred with ribothymidine and thymidine. Mouse antibodies induced by m7G-hemocyanin showed the highest specificity. They also recognized m7G in the isolated mRNA cap structure m7G(5')ppp(5')A.