Investigations on the expression of cytokines in the canine corpus luteum in relation to dioestrus

Control of luteal regression in the dog is still poorly understood. Unlike other domestic animal species, luteolysis is not prevented by hysterectomy. Indications that PGF(2alpha) may act as an endogenous luteolytic agent have been found only in pregnant animals during the prepartal decline of progesterone. Evidence from several species indicates that the immune system plays an important role in corpus luteum (Cl) function, possibly by the release of cytokines from immigrant immune cells. Hence, in the present experiment we attempted to examine the expression of cytokines in the canine Cl during the course of dioestrus (formation and regression of the Cl), using RT-PCR. Groups of 4-5 bitches were ovario-hysterectomised on days 5, 15, 25, 35, 45 and 60-80 after ovulation. Canine-specific primers for IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNFalpha, IFNgamma and TGFbeta1 were used. Positive and negative controls were included in all tests. Adequate expression was confirmed by sequencing selective samples of positive PCR products. The expression of mRNA for IL-8, IL-10, IL-12, TNF-alpha and TGF-beta1 was detected in all samples at each stage of dioestrus, without any obvious variations indicating a likely modulatory function of these cytokines in differentiation, maintenance or regression of the canine Cl. All tests for the expression of mRNA for IL-4, IL-1beta and IL-2 were negative. More negative than positive results were obtained when testing for the expression of mRNA for IL-6 and IFN-gamma, leading to the conclusion that expression of these two cytokines is at a low level, and no conclusion can be drawn as to their involvement in control of Cl function.     

Three-dimensional structure of a macromolecular assembly that regulates type III secretion in Yersinia pestis

Yersinia pestis, the causative agent of plague, utilizes a type III secretion system (T3SS) to inject effector proteins directly into the cytosol of mammalian cells where they interfere with signal transduction pathways that regulate actin cytoskeleton dynamics and inflammation, thereby enabling the bacterium to avoid engulfment and destruction by macrophages. Type III secretion normally does not occur in the absence of close contact with eukaryotic cells. Negative regulation is mediated in part by a multiprotein complex that has been proposed to act as a physical impediment to type III secretion by blocking the entrance to the secretion apparatus prior to contact with mammalian cells. This complex is composed of YopN, its heterodimeric secretion chaperone SycN-YscB, and TyeA. Here, we report two crystal structures of YopN in complex with its heterodimeric secretion chaperone SycN-YscB and the co-regulatory protein TyeA, respectively. By merging these two overlapping structures, it was possible to construct a credible theoretical model of the YopN-SycN-YscB-TyeA complex. The modeled assembly features the secretion signaling elements of YopN at one end of an elongated structure and the secretion regulating TyeA binding site at the other. A patch of highly conserved residues on the surface of the C-terminal alpha-helix of TyeA may mediate its interaction with structural components of the secretion apparatus. Conserved arginine residues that reside inside a prominent cavity at the dimer interface of SycN-YscB were mutated in order to investigate whether they play a role in targeting the YopN-chaperone complex to the type III secretion apparatus. One of the mutants exhibited a phenotype that is consistent with this hypothesis.     

Functions and pathophysiological roles of phospholipase D in the brain

Ten years after the isoforms of mammalian phospholipase D (PLD), PLD1 and 2, were cloned, their roles in the brain remain speculative but several lines of evidence now implicate these enzymes in basic cell functions such as vesicular trafficking as well as in brain development. Many mitogenic factors, including neurotransmitters and growth factors, activate PLD in neurons and astrocytes. Activation of PLD downstream of protein kinase C seems to be a required step for astroglial proliferation. The characteristic disruption of the PLD signaling pathway by ethanol probably contributes to the delay of brain growth in fetal alcohol syndrome. The post-natal increase of PLD activities concurs with synapto- and myelinogenesis in the brain and PLD is apparently involved in neurite formation. In the adult and aging brain, PLD activity has antiapoptotic properties suppressing ceramide formation. Increased PLD activities in acute and chronic neurodegeneration as well as in inflammatory processes are evidently due to astrogliosis and may be associated with protective responses of tissue repair and remodeling. ARF-regulated PLD participates in receptor endocytosis as well as in exocytosis of neurotransmitters where PLD seems to favor vesicle fusion by modifications of the shape and charge of lipid membranes. Finally, PLD activities contribute free choline for the synthesis of acetylcholine in the brain. Novel tools such as RNA interference should help to further elucidate the roles of PLD isoforms in brain physiology and pathology.     

Thio wax ester biosynthesis utilizing the unspecific bifunctional wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase of Acinetobacter sp. strain ADP1

The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5'His(6)WS/DGAT comprising an N-terminal His(6) tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5'His(6)atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein(-1) min(-1) was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5'His(6)WS/DGAT with 1-hexadecanethiol and palmitoyl-CoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1OmegaKm, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1OmegaKm in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.     

Axon guidance: opposing EPHects in the growth cone

Cells communicate with other cells via (trans) interaction between membrane-linked ephrins and Eph receptors. In this issue of Cell, Pfaff and colleagues (Marquardt et al., 2005) demonstrate that coexpressed ephrin-As and Ephs do not interact in cis but rather segregate into separate membrane domains, from which they signal opposing effects during motor axon guidance.     

Influence of high-intensity exercise training and anabolic androgenic steroid treatment on rat tissue glycogen content

To increase tissue glycogen content many athletes use anabolic androgenic steroids (AAS). However, the literature concerning the effects of androgens on glycogen metabolism is conflicting. This study aimed to determine the influence of training and AAS on body weight (bw), triglycerides, glucose, tissue glycogen and transaminases levels. Male Wistar rats, randomized into four groups (sedentary vehicle (SV), sedentary AAS (SA), trained vehicle (TV) and trained AAS (TA)), were treated with nadrolone (5 mg/Kg, 2x/week, i.m.) or vehicle. Trained rats performed jumps into water (4 sets, 10 repetitions, 30 sec rest) carrying a 50-70% body wt-load strapped to the chest (5 days/week,6 weeks). Two days after the last session, the animals were killed (bifatorial ANOVA+Tukey test; P < 0.05). Trained animals presented lower bw (TV:345+/-7 vs. SV:380+/-7 and TA:328+/-4 vs SA:370+/-11 g) and triglycerides levels (TV:77+/-3 vs. SV:98+/-4 and TA:79+/-3 vs. SA:98+/-8 mg/dL) and higher glycogen content in liver (TV:5.3+/-0.2 vs. SV:3.9+/-0.1 and TA:5.3+/-0.3 vs. SA:4.6+/-0,2 mg/100 mg) and in gastrocnemious (TV:0.70+/-0.02 vs. SV:0.49+/-0.01 and TA:0.73+/-0.03 vs. SA:0.57+/-0.02 mg/100 mg) than sedentary ones. In the cardiac muscle, the association between training and AAS increased glycogen content (TA:0.19+/-0.01 > SV:0.13+/-0.01=TV:0.13+/-0.01=SA:0.14+/-0.01 mg/100 mg). In the soleus AAS increased glycogen (SA:0.53+/-0.03 vs. SV:0.43+/-0.01 and TA:0.58+/-0.02 vs. TV:0.48+/-0.01 mg/100 mg). Exercise training and AAS had no effect on blood glucose and transaminases levels. Training and AAS effects on glycogen supercompensation are tissue-dependent and the effects of association between them were only observed in the cardiac muscle. These data emphasize the necessity of more studies to confirm greater effects of AAS than those promoted by physical exercise.     

Floral scent emission and pollinator attraction in two species of <Emphasis Type="Italic">Gymnadenia</Emphasis> (Orchidaceae)

We investigated scent composition and pollinator attraction in two closely related orchids, Gymnadenia conopsea (L.) R.Br. s.l. and Gymnadenia odoratissima (L.) Rich. in four populations during the day and night. We collected pollinators of both species using hand nets and sampled floral odour by headspace sorption. We analysed the samples by gas chromatography with mass spectrometry to identify compounds and with electroantennographic detection to identify compounds with physiological activity in pollinators. In order to evaluate the attractiveness of the physiologically active compounds, we carried out trapping experiments in the field with single active odour substances and mixtures thereof. By collecting insects from flowers, we caught eight pollinators of G. conopsea, which were members of four Lepidoptera families, and 37 pollinators of G. odoratissima, from five Lepidopteran families. There was no overlap in pollinator species caught from the two orchids using nets. In the scent analyses, we identified 45 volatiles in G. conopsea of which three (benzyl acetate, eugenol, benzyl benzoate) were physiologically active. In G. odoratissima, 44 volatiles were identified, of which seven were physiologically active (benzaldehyde, phenylacetaldehyde, benzyl acetate, 1-phenyl-2,3-butandione, phenylethyl acetate, eugenol, and one unknown compound). In field bioassays using a mixture of the active G. odoratissima compounds and phenylacetaldehyde alone we caught a total of 25 moths, some of which carried Gymnadenia pollinia. A blend of the active G. conopsea volatiles placed in the G. odoratissima population did not attract any pollinators. The two orchids emitted different odour bouquets during the day and night, but G. odoratissima showed greater temporal differences in odour composition, with phenylacetaldehyde showing a significant increase during the night. The species differed considerably in floral odour emission and this differentiation was stronger in the active than non-active compounds. This differentiation of the two species, especially in the emission of active compounds, appears to have evolved under selection for attraction of different suites of Lepidopteran pollinators.     

Association between tree-ring and needle δ<Superscript>13</Superscript>C and leaf gas exchange in <Emphasis Type="Italic">Pinus halepensis</Emphasis> under semi-arid conditions

Associations between δ¹³C values and leaf gas exchanges and tree-ring or needle growth, used in ecophysiological compositions, can be complex depending on the relative timing of CO₂ uptake and subsequent redistribution and allocation of carbon to needle and stem components. For palaeoenvironmental and dendroecological studies it is often interpreted in terms of a simple model of δ¹³C fractionation in C₃ plants. However, in spite of potential complicating factors, few studies have actually examined these relationships in mature trees over inter- and intra-annual time-scales. Here, we present results from a 4 years study that investigated the links between variations in leaf gas-exchange properties, growth, and dated δ¹³C values along the needles and across tree rings of Aleppo pine trees growing in a semi-arid region under natural conditions or with supplemental summer irrigation. Sub-sections of tissue across annual rings and along needles, for which time of formation was resolved from growth rate analyses, showed rapid growth and δ¹³C responses to changing environmental conditions. Seasonal cycles of growth and δ¹³C (up to ~4‰) significantly correlated (P<0.01) with photosynthetically active radiation, vapour pressure deficit, air temperature, and soil water content. The irrigation significantly increased leaf net assimilation, stomatal conductance and needle and tree-ring growth rate, and markedly decreased needle and tree-ring δ¹³C values and its sensitivity to environmental parameters. The δ¹³C estimates derived from gas-exchange parameters, and weighted by assimilation, compared closely with seasonal and inter-annual δ¹³C values of needle- and tree-ring tissue. Higher stomatal conductances of the irrigated trees (0.22 vs. 0.08 mol m⁻² s⁻¹ on average) corresponded with ~2.0‰ lower average δ¹³C values, both measured and derived. Derived and measured δ¹³C values also indicated that needle growth, which occurs throughout the stressful summer was supported by carbon from concurrent, low rate assimilation. For Aleppo pine under semi-arid and irrigated conditions, the δ¹³C of tree-ring and needle material proved, in general, to be a reasonable indicator of integrated leaf gas-exchange properties.