Crystallization of recombinant rat cathepsin B

A glycosylation-minus mutant of rat cathepsin B expressed in yeast has been purified and crystallized. X-ray diffraction data have been collected and molecular replacement for solving the structure is in progress. The space group for the recombinant rat cathepsin B was determined to be P2(1) with unit cell dimensions alpha = 62.2 A, b = 90.19 A, c = 47.07 A, and beta = 97.43 degrees. A unit cell contains 4 molecules and 2 molecules per asymmetric unit.     

High-level production of human alpha- and beta-globins in insect cells.

High-level production of human alpha- and beta-globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. Two recombinant baculoviruses for alpha-globin, H alpha and H beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mRNA coding region in H beta alpha. This insertion results in a 40% increase in yield of alpha-globin over that of H alpha. Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both alpha- and beta-globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.     

Postimplantation development of mitomycin C-treated mouse blastocysts

Treatment of morula-stage mouse embryos with mitomycin C (0.004-0.5 microgram/ml) in vitro resulted in a decrease in the number of inner cell mass (ICM) cells at the blastocyst stage. The trophectoderm population was reduced only at the highest dosage (0.5 microgram/ml) tested. Postblastocyst development in vitro was retarded: Fewer embryos formed trophoblastic outgrowth, and the ICM was poorly developed. The embryo transfer experiments demonstrated that a reduction in ICM cell numbers diminished the potential of embryogenesis. The presence of a sufficient number of trophoblasts and ICM cells in the blastocyst is therefore a prerequisite for successful implantation and embryogenesis. The mitomycin-treated blastocysts with only 70% of normal ICM cells developed to egg cylinders that were about half normal size, but by days 12-14 the body size of the surviving embryo was similar to that of the control embryo. Morphogenesis was retarded during the early organogenesis stages, but only a slight delay was seen in the treated embryo on day 12. Such observation strongly suggests that a restorative phase of growth and morphogenesis has occurred during the immediate postimplantation period.     

Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family

We have isolated from a constructed lambda gt11 expression library two classes of cDNA clones encoding the entire sequence of the maize GSH S-transferases GST I and GST III. Expression of a full-length GST I cDNA in E. coli resulted in the synthesis of enzymatically active maize GST I that is immunologically indistinguishable from the native GST I. Another GST I cDNA with a truncated N-terminal sequence is also active in heterospecific expression. Our GST III cDNA sequence differs from the version reported by Moore et al. [Moore, R. E., Davies, M. S., O'Connell, K. M., Harding, E. I., Wiegand, R. C., and Tiemeier, D. C. (1986) Nucleic Acids Res. 14:7227-7235] in eight reading frame shifts which result in partial amino acid sequence conservation with the rat GSH S-transferase sequences. The GST I and GST III sequences share approximately 45% amino acid sequence homology. Both the GST I and the GST III mRNAs contain different repeating motifs in front of the initiation codon ATG. Multiple poly(A) addition sites have been identified for these two classes of maize GSH S-transferase messages. Genomic Southern blotting results suggest that both GST I and GST III are present in single or low copies in the maize (GT112 RfRf) genome.     

Effect of 5-isosorbide mononitrate on isosorbide dinitrate-induced relaxation of rabbit aortic rings

The ability of isosorbide dinitrate (ISDN) and its two metabolites, 5-isosorbide mononitrate (5-ISMN) and 2-isosorbide mononitrate (2-ISMN), to relax phenylephrine-contracted rabbit aortic rings was compared. The three organic nitrates demonstrated similar efficacy. ISDN was found to be the most potent (median effective dose (ED50); 1.5 X 10(-7) +/- 1.1 X 10(-7) M), followed by 2-ISMN (ED50, 1.8 X 10(-6) +/- 9 X 10(-7) M) and 5-ISMN (ED50, 8.2 X 10(-6) +/- 3.6 X 10(-6) M). The log dose-response curve of ISDN in rabbit aortic rings was constructed in the absence and presence of three fixed concentrations of 5-ISMN (5 X 10(-6), 10(-5), and 3 X 10(-5) M). No shift in the ISDN dose-response curve at high ISDN concentrations was noted in the presence of 5-ISMN. Using the isobolographic method with fixed ISDN/5-ISMN ratio mixtures, no evidence for an antagonistic effect of 5-ISMN on ISDN-induced vasodilation was obtained. Analysis of the fixed ISDN/5-ISMN ratio mixture responses by the median-effect plot showed no antagonistic effect. It is concluded that in rabbit aortic rings 5-ISMN, the major metabolite of ISDN, is not an antagonist of ISDN at a "nitrate receptor," and no support is provided for the hypothesis that the accumulation in plasma of metabolites (e.g., 5-ISMN) with longer half-lives than the parent drug explains tolerance to organic nitrates.     

Retention of apolipoprotein B and cholesterol by perfused heart during lipolysis of very-low-density lipoprotein

The fate and mechanism of removal of apolipoproteins and lipids of human very-low-density lipoproteins were determined in the perfused rat heart. Approx. 50% of the VLDL triacylglycerol was hydrolyzed during a 2 h perfusion. Phospholipid phosphorus, apolipoproteins C-II, C-III and E were quantitatively recovered in the medium. However, there was a loss of unesterified (17 +/- 6%) and esterified (19 +/- 8%) cholesterol from the perfusion medium. Apolipoprotein B was retained by the heart, as determined by the loss of immunoassayable apolipoprotein B (30 +/- 5%) or the uptake of 125I-labelled apolipoprotein of VLDL (9 +/- 2%) from the perfusion medium. The discrepancy in the two methods for estimating apolipoprotein removal was shown to be due to the modification of apolipoprotein B-containing lipoproteins, which was such that they were no longer precipitated with antibodies to apolipoprotein B. The labelled apolipoprotein B, retained by the heart, could be partially released by perfusion of the heart with buffer containing heparin (14 +/- 2%) or trypsin (50 +/- 2%). Labelled apolipoprotein uptake by the heart was reduced by 90% when lipoprotein lipase was first released by heparin or when VLDL was treated with 1,2-cyclohexanedione to modify arginine residues of apolipoproteins. Very little extensive degradation of the apoprotein to low molecular weight material occurred during the 2 h perfusion, since 95% of the tissue label was precipitated by trichloroacetic acid. It is concluded that there is retention of apolipoprotein B, cholesteryl ester and cholesterol by the perfused heart during catabolism of VLDL. The data are consistent with the concept that the retention of apolipoprotein B requires membrane-bound lipoprotein lipase or an interaction with the cell surfaces that is modified by heparin. The overall process also involves arginine residues of apolipoproteins. At least 50% of the labelled apolipoprotein retained in the tissue is associated with lipoprotein lipase and other cell surface sites, while the remainder may be taken up by the cells.     

Phylogenetic placement of the unusual jumping spider Depreissia Lessert,and a new synapomorphy uniting Hisponinae and Salticinae (Araneae,Salticidae)

The relationships of the unusual salticid spider Depreissia from central Africa and Borneo have been difficult to resolve, obscured by its highly modified ant-like body. Phylogenetic analysis of the gene 28S strongly supports its placement outside the major clade Salticinae and within the clade of cocalodines, spartaeines and lapsiines, with weaker support for a relationship with the cocalodines in particular. Excluding the genus from the Salticinae is supported also by the presence of a median apophysis on the male palp, and by the lack of a cymbial apical groove cradling the tip of embolus, which is newly presented here as a synapomorphy of Hisponinae plus Salticinae.     

Investigating Empathy-Like Responding to Conspecifics’ Distress in Pet Dogs

Empathy covers a wide range of phenomena varying according to the degree of cognitive complexity involved; ranging from emotional contagion, defined as the sharing of others’ emotional states, to sympathetic concern requiring animals to have an appraisal of the others’ situation and showing concern-like behaviors. While most studies have investigated how animals reacted in response to conspecifics’ distress, dogs so far have mainly been targeted to examine cross-species empathic responses. To investigate whether dogs would respond with empathy-like behavior also to conspecifics, we adopted a playback method using conspecifics’ vocalizations (whines) recorded during a distressful event as well as control sounds. Our subjects were first exposed to a playback phase where they were subjected either to a control sound, a familiar whine (from their familiar partner) or a stranger whine stimulus (from a stranger dog), and then a reunion phase where the familiar partner entered the room. When exposed to whines, dogs showed a higher behavioral alertness and exhibited more stress-related behaviors compared to when exposed to acoustically similar control sounds. Moreover, they demonstrated more comfort-offering behaviors toward their familiar partners following whine playbacks than after control stimuli. Furthermore, when looking at the first session, this comfort offering was biased towards the familiar partner when subjects were previously exposed to the familiar compared to the stranger whines. Finally, familiar whine stimuli tended to maintain higher cortisol levels while stranger whines did not. To our knowledge, these results are the first to suggest that dogs can experience and demonstrate “empathic-like” responses to conspecifics’ distress-calls.     

Disentangling genetic and epigenetic determinants of ultrafast adaptation

A major rationale for the advocacy of epigenetically mediated adaptive responses is that they facilitate faster adaptation to environmental challenges. This motivated us to develop a theoretical–experimental framework for disclosing the presence of such adaptation‐speeding mechanisms in an experimental evolution setting circumventing the need for pursuing costly mutation–accumulation experiments. To this end, we exposed clonal populations of budding yeast to a whole range of stressors. By growth phenotyping, we found that almost complete adaptation to arsenic emerged after a few mitotic cell divisions without involving any phenotypic plasticity. Causative mutations were identified by deep sequencing of the arsenic‐adapted populations and reconstructed for validation. Mutation effects on growth phenotypes, and the associated mutational target sizes were quantified and embedded in data‐driven individual‐based evolutionary population models. We found that the experimentally observed homogeneity of adaptation speed and heterogeneity of molecular solutions could only be accounted for if the mutation rate had been near estimates of the basal mutation rate. The ultrafast adaptation could be fully explained by extensive positive pleiotropy such that all beneficial mutations dramatically enhanced multiple fitness components in concert. As our approach can be exploited across a range of model organisms exposed to a variety of environmental challenges, it may be used for determining the importance of epigenetic adaptation‐speeding mechanisms in general.