Gene expression and molecular composition of phospholipids in rat brain in relation to dietary n-6 to n-3 fatty acid ratio

Rats were fed from conception till adulthood either with normal rat chow with a linoleic (LA) to linolenic acid (LNA) ratio of 8.2:1 or a rat chow supplemented with a mixture of perilla and soy bean oil giving a ratio of LA to LNA of 4.7:1. Fat content of the feed was 5%. Fatty acid and molecular species composition of ethanolamine phosphoglyceride was determined. Effect of this diet on gene expression was also studied. There was an accumulation of docosahexaenoic (DHA) and arachidonic acids (AA) in brains of the experimental animals. Changes in the ratio sn-1 saturated, sn-2 docosahexaenoic to sn-1 monounsaturated, sn-2 docosahexaenoic were observed. Twenty genes were found overexpressed in response to the 4.7:1 mixture diet and four were found down-regulated compared to normal rat chow. Among them were the genes related to energy household, lipid metabolism and respiration. The degree of up-regulation exceeded that observed with perilla with a ratio of LA to LNA 8.2:1 [Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2619]. It was concluded that brain sensitively reacts to the fatty acid composition of the diet. It was suggested that alteration in membrane architecture and function coupled with alterations in gene expression profiles may contribute to the observed beneficial impact of n-3 type polyunsaturated fatty acids on cognitive functions.     

Specific expression of the Drosophila midline-jumper retro-transposon in embryonic CNS midline cells

Here we describe of a novel Drosophila LTR-type retrotransposon that is expressed in the embryonic CNS midline glia and in the embryonic germ cells. The element is related to the gypsy and burdock retrotransposons and was termed midline-jumper. In addition to cDNA clones generated from internal retrotransposon sequences, we have identified one cDNA clone that appears to reflect a transposition event, indicating that the midline-jumper retrotransposon is not only transcribed but also able to transpose during Drosophila development.     

Convergence of calcium signaling pathways of pathogenic elicitors and abscisic acid in Arabidopsis guard cells

A variety of stimuli, such as abscisic acid (ABA), reactive oxygen species (ROS), and elicitors of plant defense reactions, have been shown to induce stomatal closure. Our study addresses commonalities in the signaling pathways that these stimuli trigger. A recent report showed that both ABA and ROS stimulate an NADPH-dependent, hyperpolarization-activated Ca(2+) influx current in Arabidopsis guard cells termed "I(Ca)" (Z.M. Pei, Y. Murata, G. Benning, S. Thomine, B. Klüsener, G.J. Allen, E. Grill, J.I. Schroeder, Nature [2002] 406: 731-734). We found that yeast (Saccharomyces cerevisiae) elicitor and chitosan, both elicitors of plant defense responses, also activate this current and activation requires cytosolic NAD(P)H. These elicitors also induced elevations in the concentration of free cytosolic calcium ([Ca(2+)](cyt)) and stomatal closure in guard cells. ABA and ROS elicited [Ca(2+)](cyt) oscillations in guard cells only when extracellular Ca(2+) was present. In a 5 mM KCl extracellular buffer, 45% of guard cells exhibited spontaneous [Ca(2+)](cyt) oscillations that differed in their kinetic properties from ABA-induced Ca(2+) increases. These spontaneous [Ca(2+)](cyt) oscillations also required the availability of extracellular Ca(2+) and depended on the extracellular potassium concentration. Interestingly, when ABA was applied to spontaneously oscillating cells, ABA caused cessation of [Ca(2+)](cyt) elevations in 62 of 101 cells, revealing a new mode of ABA signaling. These data show that fungal elicitors activate a shared branch with ABA in the stress signal transduction pathway in guard cells that activates plasma membrane I(Ca) channels and support a requirement for extracellular Ca(2+) for elicitor and ABA signaling, as well as for cellular [Ca(2+)](cyt) oscillation maintenance.     

The col-1 module of human matrix metalloproteinase-2 (MMP-2): structural/functional relatedness between gelatin-binding fibronectin type II modules and lysine-binding kringle domains

Human matrix metalloproteinase-2 (MMP-2) contains three in-tandem fibronectin type II (FII) repeats that bind gelatin. Here, we report the NMR solution structure of the first FII module of MMP-2 (col-1). The latter is described as a characteristic, globular FII fold containing two beta-sheets, a stretch of 3(1)-helix, a turn of alpha-helix, and an exposed hydrophobic surface lined with aromatic residues. We show that col-1 binds (Pro-Pro-Gly)6, a mimic of gelatin, with a Ka of approx. 0.42 mm(-1), and that its binding site involves a number of aromatic residues as well as Arg34, as previously found for the second and third homologous repeats. Moreover, the affinity of the in-tandem col-1+2 construct (col-12) toward the longer ligand (Pro-Pro-Gly)12 is twice that for (Pro-Pro-Gly)6, as expected from mass action. A detailed structural comparison between FII and kringle domains indicates that four main conformational features are shared: two antiparallel beta-sheets, a central 3(1)-helix, and the quasiperpendicular orientation of the two proximal Cys-Cys bonds. Structure superposition by optimizing overlap of cystine bridge areas results in close juxtaposition of their main beta-sheets and 31-helices, and reveals that the gelatin binding site of FII modules falls at similar locations and exhibits almost identical topological features to those of the lysine binding site of kringle domains. Thus, despite the minor (<15%) consensus sequence relating FII modules to kringles, there is a strong folding and binding site structural homology between the two domains, enforced by key common conformational determinants.     

Investigation of catalytic reaction with spectrofluorimetric method

Numerous derivatives of nicotinic acid hydrazids are suitable for sensitive fluorescence determination of metal ions. The reaction proceeds upon ultraviolet illumination in the presence of an oxidizing agent and catalyst forming a fluorescent product. Therefore, the catalyst metal ion can be quantitatively determined by fluorescence methods. We have studied the reaction of nicotinic acid (di-pyridin-2-yl-methylene)-hydrazide (NADPMH), catalyzed by Mn(II)ion, and determined the optimal parameters of metal ion method.     

Optimized sensitivity of allele-specific PCR for prenatal typing of human platelet alloantigen single nucleotide polymorphisms

PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.     

Scanning electron microscopic examinations on retarded bone defect healing in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats

To date, no detailed knowledge from animal experiments is available on the kind and extent of osseous and mineral metabolic disorders in genetically determined, insulin-dependent Type I diabetes. The purpose of this study was to examine the influence of the diabetic metabolic state in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats on bone defect healing. Eighty spontaneously-diabetic BB/OK rats with a blood-glucose value of 391 +/- 106 mg% (mean +/- SD) at the time of manifestation were used in the study. Based on blood-glucose values at the time of surgery (mg%), postoperative blood-glucose course (mg%) and postoperative insulin requirements (IU/kg), the animals were divided into groups with well-compensated (n = 40, 170 +/- 101 mg%; 221 +/- 120 mg%; 2.1 +/- 1 IU/kg) or poorly compensated (n = 40; 371 +/- 158 mg%; 357 +/- 83 mg%; 5.2 +/- 1.4 IU/kg) metabolic state. Forty LEW.1A rats served as the normoglycemic controls (95 +/- 18 mg%). Using a 1-mm-diameter Kirschner wire, a hole of femoral bone ca. 1 cm proximal to the knee joint space was centrally drilled. Ten animals from each group were killed on postoperative days 7, 14, 24, and 42, and specimens were taken for analysis. Using SEM to measure regions of new bone semiautomatically and quantitatively, also determining the number, area, and circumference of regions not yet filled with new bone. Up to postoperative day 14, very significant differences (p < 0.0001) for all investigated characteristics were found between the spontaneously-diabetic BB/OK rats and the control animals--in favor of the controls--and up to postoperative day 24 within the group of spontaneously-diabetic BB/OK rats, where the well-compensated animals had significantly better results in terms of number and area of regions of bone not yet filled with new bone formations. Forty-two days postoperatively, SEM observations showed no differences between examination groups. The process of bone defect healing in spontaneously-diabetic rats was disturbed only in the early phase and exhibited retardation in its progression. After 42 days, bone defect healing was complete, regardless of the diabetic metabolic state; no differences were detected with the SEM between examination groups at this time point.     

Effect of different doses of prostaglandin E2 on intrauterine pressure and uterine motility during diestrus in experimental cows

Studies in human medicine proved the important role of prostaglandin E2, which stimulates uterine contractions in vivo and in vitro and has been extensively used to ripen the cervix around labor. We wanted to demonstrate that increasing the dosage of prostaglandin E2 (1.25 mg, 2.5 mg, 5 mg and 10 mg) provokes an increase in intrauterine pressure and uterine motility in cattle. Five healthy, lactating dairy cows were used as experimental animals for this study. Intrauterine pressure was recorded during the diestrus phase (1 recording per cow and diestrus phase) by means of a transcervically placed intraluminal pressure microtransducer. Physiologic uterine motility was recorded for 30 min, then placebo or one of the prostaglandin E2- dosages was administered through an indwelling catheter in the jugular vein, followed by a 2-h recording period (eight 15-min periods). Area under the curve (AUC), mean amplitude, frequency of pressure waves and intrauterine pressure were analyzed. Furthermore, we recorded protocols for monitoring heart and respiratory rates and side effects at 9 given examination times. Significant differences were found for the AUC, the mean amplitude and the intrauterine pressure (P < or = 0.05), whereas the number of pressure waves per 15 min did not differ significantly among treatments. Peak values for AUC, mean amplitude and intrauterine pressure were found during the first 15 min after administration of 10 mg of prostaglandin E2. Dose-effect curves showed that the 2.5 mg dosage provided the optimal ratio between myometrial stimulation and undesirable side-effects.