Song Learning in Zebra Finches Exposed to a Series of Tutors during the Sensitive Phase

Young male zebra finches exposed to a series of adult males during their sensitive phase for song learning tended to learn from several of them, in contrast to those exposed to two or more tutors simultaneously. There is a tendency to learn more from later tutors, either because the memory of their elements over-writes that of earlier ones or because sensitivity is greater at this stage. No clear relation could be found between features of the song or behaviour of tutors and whether or not they were copied. In common with earlier studies, tutor song output, beyond a certain minimum was found to be unimportant.     

Isolation of DNA from yeasts

Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.     

Ovine fetal adaptations to chronically reduced urine flow: preservation of amniotic fluid volume

Mann, Stephanie E., Mark J. M. Nijland, and Michael G. Ross.Ovine fetal adaptations to chronically reduced urine flow: preservation of amniotic fluid volume. J. Appl.Physiol. 81(6): 2588-2594, 1996.Adequateamniotic fluid (AF) volume is maintained by a balance of fetal fluidproduction (lung liquid and urine) and resorption (swallowing andintramembranous flow). Because fetal urine is the principle source ofAF, alterations in urine flow and composition directly impact AFdynamics. Intra-amniotic 1-desamino-8-D-argininevasopressin (DDAVP) is rapidly absorbed into fetal plasma and induces amarked fetal urinary antidiuresis. To examine the effect ofintra-amniotic- DDAVP-induced fetal urinary responses on AF volume andcomposition, six chronically prepared ewes with singleton fetuses(gestation 128 ± 2 days) were studied for 72 h after a singleintra-amniotic DDAVP (50-µg) injection. After DDAVP, fetal urineosmolality significantly increased at 2 h (157 ± 13 to 253 ± 21 mosmol/kg) and remained elevated at 72 h (400 ± 13 mosmol/kg). Urinary sodium (33.0 ± 4.5 to 117.2 ± 9.7 meq/l)and chloride (26.0 ± 2.8 to 92.4 ± 8.1 meq/l) concentrations similarly increased. AF osmolality increased (285 ± 3 to 299 ± 4 mosmol/kgH₂O), although there was no change in fetalplasma osmolality (294 ± 2 mosmol/kg). Despite a 50% reductionin fetal urine flow (0.12 ± 0.03 to 0.05 ± 0.02 ml ^· kg^{1 ·} min¹at 2 h and 0.06 ± 0.01 ml ^· kg^{1 ·} min¹after 72 h), AF volume did not change (693 ± 226 to 679 ± 214 ml). There were no changes in fetal arterial blood pressures, pH,PCO₂, orPO₂ after DDAVP. We conclude the following. 1)Intra-amniotic DDAVP injection induces a prolonged decrease in fetalurine flow and increases in urine and AF osmolalities. 2) Despite decreased urine flow, AFvolume does not change. We speculate that, in response to DDAVP-inducedfetal oliguria, reversed intramembranous flow (from isotonic fetalplasma to hypertonic AF) preserves AF volume.

     

Stress activated cytokines and the heart

The ability of myocardium to successfully compensate for, and adapt to, stress ultimately determines whether the heart will decompensate and fail, or whether it will instead maintain preserved function. Despite the importance of the myocardial response to environmental stress, very little is known with respect to the biochemical mechanisms that are responsible for mediating and integrating the stress response in the heart. In the present review we will summarize recent experimental material which suggests that cytokines that are expressed within the myocardium in response to a environment injury, namely tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6), may play an important role in initiating and integrating homeostatic responses within the heart. However, these ‘stress-activated’ cytokines all have the potential to produce cardiac decompensation when expressed at sufficiently high concentrations. Accordingly, the theme that will emerge from this discussion is that the short-term expression of stress-activated cytokines within the heart may provide the heart with an adaptive response to stress, whereas long-term expression of these molecules may be frankly maladaptive by producing cardiac decompensation.     

Effect on lipoprotein profile of replacing butter with margarine in a low fat diet: randomised crossover study with hypercholesterolaemic subjects.

OBJECTIVE--To examine the effect on lipid and lipoprotein concentrations when butter or an unsaturated margarine is used for cooking or spreading in a reduced fat diet. DESIGN--Randomised crossover study with two intervention periods of six weeks'' duration separated by a five week washout. SETTING--Community setting in New Zealand. SUBJECTS--49 volunteers with polygenic hypercholesterolaemia and baseline total cholesterol concentration in the range 5.5-7.9 mmol/l. MAIN OUTCOME MEASURES--Concentrations of total and low density lipoprotein, Lp(a) lipoprotein, high density lipoprotein, apolipoprotein B 100, and apolipoprotein A I. RESULTS--Concentrations of low density lipoprotein cholesterol and apolipoprotein B were about 10% lower with margarine than with butter. Lp(a) lipoprotein and high density lipoprotein cholesterol concentrations were similar with the two diets. CONCLUSION--Despite concerns about adverse effects on lipoproteins of trans fatty acids in margarines, the use of unsaturated margarine rather than butter by hypercholesterolaemic people is associated with a lipoprotein profile that would be expected to reduce cardiovascular risk.     

A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histi-dine tag and an enterokinase cleavage site exhibits     

Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast

Background Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28–cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis.Results The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55Δ cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids.Conclusions We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.     

Identification of Homeotic Target Genes in Drosophila Melanogaster Including Nervy, a Proto-Oncogene Homologue

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.     

The application of robotics and mass spectrometry to the characterisation of the Drosophila melanogaster indirect flight muscle proteome

The rapid accumulation of sequence data generated by the various genome sequencingprojects and the generation of expressed sequence tag databases has resulted in the need forthe development of fast and sensitive methods for the identification and characterisation oflarge numbers of gel electrophoretically separated proteins to translate the sequence data intobiological function. To achieve this goal it has been necessary to devise new approaches toprotein analysis: matrix-assisted laser desorption and electrospray mass spectrometry havebecome important protein analytical tools which are both fast and sensitive. When combinedwith a robotic system for the in-gel digestion of electrophoretically separated proteins, itbecomes possible to rapidly identify many proteins by searching databases with MS data. Thepower of this combination of techniques is demonstrated by an analysis of the proteins presentin the myofibrillar lattice of the indirect flight muscle of Drosophila melanogaster. Theproteins were separated by SDS-PAGE and in-gel proteolysis was performed bothautomatically and manually. All 16 major proteins could quickly be identified by massspectrometry. Although most of the protein components were known to be present in theflight muscle, two new components were also identified. The combination of methodsdescribed offers a means for the rapid identification of large numbers of gel separatedproteins.