Experience-dependent development of the adult optic lobe and central brain in Drosophila melanogaster.

The optic lobes, mushroom bodies (MB) and central complex (CC) of Drosophila melanogaster were investigated in order to find out whether rearing in different light regimes affect their size. Flies raised in constant light up for up to four days post eclosion had a lamina that was about 30% larger than in flies kept in constant darkness. A volume difference between light- and dark-reared flies could also be observed for the lobula plate, the MBs, and the CC. When the flies were kept in the dark for the first 12 hours of their adult life and then brought back to constant light for the next 3.5 days, the lamina was as small as the laminae of flies raised for four days in constant darkness. This finding suggests a critical period for lamina development during day one of the imago. Mutant studies suggest that the molecular mechanisms underlying this experience-dependent development might be quite diverse. For example, the structural plasticity of the mushroom bodies is abolished in the dunce mutation, whereas the light-dependent growth of the lamina is not. Finally, studies of optomotor behavior indicate an adaptational role for the structural plasticity in the optic lobe. Surprisingly, dark-reared flies see better under low light conditions than their light-reared counterparts. This suggests that a small lamina is not a bad lamina in the sense that dark-reared animals see worse. They rather adapt to the specific light-conditions they were growing up in.     

Molecular changes in the sarcoplasmic reticulum calcium ATPase during catalytic activity. A Fourier transform infrared (FTIR) study using photolysis of caged ATP to trigger the reaction cycle

Fourier transform infrared spectroscopy was used to study ligand binding and conformational changes in the Ca2(+)-ATPase of sarcoplasmic reticulum. Novel in infrared difference spectroscopy, the catalytic cycle in the IR sample was started by photolytic release of ATP from an inactive, photolabile ATP-derivative (caged ATP). Small, but characteristic infrared absorbance changes were observed upon ATP release. On the basis of model spectra, the absorbance changes corresponding to the trigger and substrate reactions, i.e. to photolysis of caged ATP and hydrolysis of ATP, were separated from the absorbance changes due to the active ATPase reflecting formation of the phosphorylated Ca2E1P enzyme form. A major rearrangement of ATPase conformation as the result of catalysis can be excluded.     

Antisense RNA decreases AP33 gene expression and cytoadherence by <Emphasis Type="Italic">T. vaginalis</Emphasis>

Background  

Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach.     

Involvement of kil and kor genes in the phenotype of a host-range mutant of RP4

Summary Plasmid pRP761 is a derivative of the promiscuous plasmid RP4, which has a Tn76 insert 1.8 kb from its EcoRI site within the trfB region (Barth 1979). This mutation was pleiotropic, having three effects: the plasmid is unstably maintained in E. coli, it reduces the growth rate of its host and it has suffered a reduction in host-range. We show that pRP761 has reduced expression from both its korA and korB genes and that Tn76 has inserted between them. Fragment exchange experiments showed that this is the only mutant region in pRP761 and is therefore solely responsible for the pleiotropic effects. A spontaneous deletion derivative pRP761-6 has lost Tn76 and its adjacent kilA and korA genes: it has reacquired stability, does not inhibit host growth but is still reduced in its host-range. The provision of cloned korA ⁺ in trans complements the first two phenotypic effects in pRP761 to a large extent, but neither korA ⁺ alone nor korA ⁺ with korB ⁺ complements the host-range reduction in pRP761 or pRP761-6. A possible explanation for these results is that there is a site between korA and korB, affected by the Tn76 insert, that is essential to stable replication of these plasmids in some of their bacterial hosts.     

Continuous infusion interleukin-2 and tumor-derived activated cells as treatment of advanced solid tumors: a National Biotherapy Study Group Trial

Metastases from patients with solid tumors were harvested from 196 patients for the purpose of growing tumor-derived activated cells (TDAC). Cells were prepared from autologous tumor cultures by incubation with Interleukin-2 (IL-2) followed by repeated exposure to tumor antigen and/or anti-CD3 monoclonal antibody. Initial growth success was achieved in 66%; 45/56 (80%) of these early cultures were subsequently expanded for in vivo therapy. It took a mean of 69.4 +/- 24.0 days to grow TDAC for treatment. Thirty-eight patients were treated with cyclophosphamide (1 g/m2) on day one followed by a 96-hour continuous infusion of IL-2 (18 x 10(6) IU/m2/day) on days 2-5 and approximately 10(11) TDAC on day 2. Patients subsequently received monthly IL-2 as a 96-hour constant infusion if their cancers were stable or regressing. Median age was 51 yrs; 58% were male. Performance status was 0-1 in 64%, 29% had lung metastases; 34% had liver metastases. The usual IL-2 toxicities were seen. Responses were seen only in 1/38 patients (3%); a partial response in a patient with lymphoma. Forty-two percent were stable 90 days post-treatment, the rest were progressive or inevaluable. We conclude that a treatment plan for IL-2/TDAC is technically difficult, costly, and not practical under these conditions. Clinical results to date are not clearly different than those obtained with other IL-2 regimens.     

Reactions between dipeptidyl peptidase IV and diacyl hydroxylamines: mechanistic investigations

Kinetics of inactivation of dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) by N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines and their enzyme-catalyzed hydrolysis were followed using independent monitoring methods, all giving similar efficiency ratios of Kcat/Kinact. Different temperature dependences of the DP IV-inactivation and enzyme-catalyzed hydrolysis provide evidence of independent rate determining steps for both reactions. Activation parameters of inactivation are similar to those of spontaneous decomposition of the compounds, suggesting a mechanistic relationship. Investigation of DP IV-inactivation, DP IV-catalyzed hydrolysis of N-Ala-Pro-O-Bz(4-NO2) and the decomposition of the suicide substrate in H2O and D2O gave solvent isotope effects of 4.65, 2.54 and 1.02, respectively. A proton inventory of the inactivation reaction indicates involvement of more than one proton in the formation or breakdown of its transition state. The linear proton inventory found for the hydrolytic reaction is consistent with one proton transition in the rate determining step and resembles the rate limiting deacylation of Ala-Pro-DP IV. The hypothetical reaction model now locates splitting in both reactions prior to formation of a covalent intermediate during the catalytic cycle.     

Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.     

The effect of cyclophosphamide and isoniazid (INH) alone and in combination on the centromere separation sequence in chinese hamster bone marrow cells

Summary X-irradiation of Chinese hamsters with 50 and 200 rad delayed the centromere separation of bone marrow chromosomes. Combination of a treatment with 13.3 mg cyclophosphamide/kg body wt. and 50 rad did not result in an enhanced effect compared with the single treatments. Again, the separation sequence was not influenced. No delay was induced by a treatment with 125 mg isoniazid/kg, but a slightly and significantly earlier separation of the chromatids in both groups of metacentric and acrocentric chromosomes occurred. The separation delay seen after irradiation with 50 rad disappeared when the animals were pretreated with isoniazid. Independently of these results the asynchronous separation sequence was not altered in any of the experiments.