Cut-off values for normal sperm morphology and toxicology for automated analysis of rat sperm morphology and morphometry

We used automated sperm morphology analysis to investigate rat sperm morphometry and morphology in Sprague-Dawley and Wistar rats in three research centers to develop normal baseline values for sperm morphometry and to quantify the percentage of morphologically normal sperm in healthy rats. The participating centers were IRSN in Paris, France (Sprague-Dawley rats), University of the Western Cape, South Africa (Wistar rats) and Stellenbosch University (Wistar rats), South Africa. All three centers used identical sperm isolation techniques from the cauda epididymis, the same staining protocols, identical computer-aided sperm morphometry analysis (CASMA) software and microscopes with similar optics. With CASMA, fully automated analysis of the different parts of stained sperm, e.g., head, acrosome, mid-piece, can be performed, many sperm morphometric features can be measured accurately and eventually normal sperm morphology can be defined. We found that it is possible to distinguish sperm morphometric characteristics of Sprague-Dawley and Wistar rats. We also developed cut-off values for evaluating the percentage normal sperm in these two rat strains using the automatic analysis mode. Normal sperm morphology varied between 67 and 74% by contrast with previous findings of > 90%.     

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [{"type":"entrez-nucleotide","attrs":{"text":"KF611679","term_id":"560187972","term_text":"KF611679"}}KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.     

Impact of Anti-Peroxynitrite-Damaged-Thymidine- Monophosphate Antibodies on Disease Activity in Patients with Systemic Lupus Erythematosus

Present study probes the role of peroxynitrite (ONOO^-)-modified thymidine-5′-monophosphate (TMP) in SLE patients with different disease activity scores according to the SLE Disease Activity Index (SLEDAI). Serum analysis showed significant increased number of subjects positive for anti-ONOO^--TMP-protein antibodies in SLE patients with different SLEDAI scores. Interestingly, the levels of these antibodies were significantly higher among SLE patients, whose SLEDAI scores were ≥20. In addition, a significant correlation was observed between the levels of anti-ONOO^--TMP-protein antibodies and the SLEDAI score (r = 0.595, p < 0.0001). In short, this study shows a positive association between anti-ONOO^--TMP-protein antibodies and SLEDAI. The stronger response observed in patients with higher SLEDAI scores suggests that anti-ONOO^--TMP-protein antibodies may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.     

Mapping of Defense Response Gene Homologs and Their Association with Resistance Loci in Maize

Defense response genes in higher plant species are involved in a variety of signal transduction pathways and biochemical reactions to counterattack invading pathogens. In this study, a total of 366 non-redundant defense response gene homologs （DRHs）, including 124 unigenes/expressed sequence tags, 226 tentative consensuses, and 16 DRH contigs have been identified by mining the Maize Genetics and Genomics and The Institute for Genomic Research maize databases using 35 essential defense response genes. Of 366 DRHs, 202 are mapped to 152 loci across ten maize chromosomes via both the genetic and in silico mapping approaches. The mapped DRHs seem to cluster together rather than be evenly distributed along the maize genome. Approximately half of these DHRs are located in regions harboring either major resistance genes or quantitative trait loci （QTL）. Therefore, this comprehensive DRH linkage map will provide reference sequences to identify either positional candidate genes for resistance genes and/or QTLs or to develop makers for fine-mapping and marker-assisted selection of resistance genes and/or QTLs.     

Rating of perceived exertion as a tool for prescribing and self regulating interval training: a pilot study

The aim of the present study was to analyse the usefulness of the 6-20 rating of perceived exertion (RPE) scale for prescribing and self-regulating high-intensity interval training (HIT) in young individuals. Eight healthy young subjects (age = 27.5±6.7 years) performed maximal graded exercise testing to determine their maximal and reserve heart rate (HR). Subjects then performed two HIT sessions (20 min on a treadmill) prescribed and regulated by their HR (HR: 1 min at 50% alternated with 1 min at 85% of reserve HR) or RPE (RPE: 1 minute at the 9-11 level [very light-fairly light] alternated with 1 minute at the 15-17 level [hard-very hard]) in random order. HR response and walking/running speed during the 20 min of exercise were compared between sessions. No significant difference between sessions was observed in HR during low- (HR: 135±15 bpm; RPE: 138±20 bpm) and high-intensity intervals (HR: 168±15 bpm; RPE: 170±18 bpm). Walking/running speed during low- (HR: 5.7±1.2 km · h⁻¹; RPE: 5.7±1.3 km · h⁻¹) and high-intensity intervals (HR: 7.8±1.9 km · h⁻¹; RPE: 8.2±1.7 km · h⁻¹) was also not different between sessions. No significant differences were observed in HR response and walking/running speed between HIT sessions prescribed and regulated by HR or RPE. This finding suggests that the 6-20 RPE scale may be a useful tool for prescribing and self-regulating HIT in young subjects.     

Saturation mapping of the apple scab resistance gene Vf using AFLP markers

Using the amplified fragment length polymorphism (AFLP) technique combined with a ”narrow-down” bulk segregant strategy enabled us to quickly identify 15 tightly linked AFLP markers to the Vf gene that confers resistance to the apple scab disease. High-resolution mapping placed all 15 AFLP markers within an interval of 0.6 cM around the Vf region; 7 of them were found to be inseparable from the Vf gene, 1 was localized left of, and the remaining 7 located right of the Vf gene. In addition, eight previously identified RAPD markers were also mapped, but only three, including M18, AM19, and AL07, were localized within this short interval, and none co-segregated with the Vf gene. The saturation of the Vf region with AFLP markers will accelerate both marker-assisted selection and map-based cloning. The advantages of this ”narrow-down” strategy, estimation of physical distances among AFLP markers, and their potential application are also discussed. Received: 22 December 1999 / Accepted: 25 March 2000     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Effects of source of macronutrients and plant growth regulator concentrations on shoot proliferation of Cornus florida

Nodal stem segments of flowering dogwood (Cornus florida L.) were cultured on media containing seven different sources of macronutrients including full- and half-strength Murashige & Skoog (MS) macrosalts, N6, Anderson's (AND), Quoirin & Lepoivre's (LP), Nitsch & Nitsch (NIT), and Woody Plant Medium (WPM). All media contained MS micronutrients, Staba vitamins, 20 g l^-1 sucrose, and 6.5 g l^-1 Difco Bacto-agar, and were supplemented with 2.2 M 6-benzyladenine (BA) and 0.49 M indole-3-butyric acid (IBA). Following three subcultures, the best shoot proliferation was supported on media containing WPM macronutrients. To optimize the proliferation rate, shoots were cultured on WPM macronutrients supplemented with eight combinations of BA and IBA, and 3.3 M BA without IBA was determined to be the best.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid     

Cytochemical Localization of Plasma Membrane Enzyme Markers During Interiorization of Tachyzoites of Toxoplasma gondii by Macrophages

The enzyme activity of Mg-ATPase, Na⁺-K⁺-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected at the ultrastructural level in mouse peritoneal macrophages infected with untreated and with specific antibody-coated Toxoplasma gondii tachyzoites. The Mg⁺⁺-ATPase and 5'-nucleotidase were distributed throughout the macrophages'plasma membrane but were not observed in the membrane lining endocytic vacuoles containing ingested parasites; however, Na⁺-K⁺-ATPase activity was detected in the macrophages'plasma membrane as well as in the parasitophorous vacuoles that contained untreated or specific antibody-coated parasites. Reaction product, indicative of NAD(P)H-oxidase, was detected in the parasitophorous vacuoles that contained only-specific antibody-coated parasites.