Homolog of FlhDC, a master regulator for flagellum synthesis: required for pathogenicity in Erwinia carotovora subsp. carotovora

 Erwinia carotovora subsp. carotovora (Ecc) is a causal agent of soft-rot diseases in a wide variety of plants. Here, we have isolated nonmotile mutants in Ecc by in vivo insertional mutagenesis using a transposon Tn5. The sequence disrupted by the Tn5 insertion in YMU1 and YMU5 mutants was highly homologous to that of flhC and flhD genes, respectively. They are involved in the initiation of the expression of flagellum-related genes in many gram-negative bacteria such as Escherichia coli and Salmonella. With electron microscopy, the flhC and the flhD homolog mutants were shown to be aflagellate. Furthermore, the virulence of these mutants was greatly reduced in Chinese cabbage and potato compared to that of the parental strain. These results suggest that flagellar formation is required for the pathogenicity of Ecc. Received: November 5, 2002 / Accepted: December 2, 2002 Acknowledgments This research was supported in part by Grant-in-Aid (12052210) and by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (13073).     

Kurokura Solution as Immobilizing Medium for Spermatozoa of Tench (Tinca tinca L.)

This study tested KUROKURA solution (Kurokura et al., 1984, Aquaculture 37, 267–274) and its modifications (by increasing NaCl content to 160, 180 and 200 mM) on immobilizing properties for sampling and short-term preservation of potential motility of tench spermatozoa. The immobilizing solution is used because, when collected, the sperm of most samples is contaminated by urine, causing spermatozoa to be of poor quality, with low motilities and velocities (almost 0), thus resulting in a worsened fertilization and hatching rate. Sperm was sampled with a syringe containing an immobilizing solution (IS), allowing an IS:sperm ratio of 2:1, under aerobic conditions at 0–4°C. This sperm solution was stored for 10 h and untreated sperm was collected prior to fertilization as a control. Spermatozoa quality was evaluated for the cell motility and velocity parameters and also for fertilization ability and hatching rate. Results obtained for tench sperm motility, velocity, fertilization and hatching rate showed that only sperm collected in the various immobilizing solutions can be successfully used for artificial insemination and preservation after 10 h at 0–4°C. The best immobilizing solution was found to be KUROKURA 180 (180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl₂· 2H₂O and 2.38 mM NaHCO₃), giving a fertility and hatching rate of 41%, with no change in rates after 10 h storage of sperm. Control sperm without immobilizing solution showed a fertilization and hatching rate of only 6–7%.     

Ionic composition and osmolality of paddlefish (Polyodon spathula, Acipenseriformes) seminal fluid

Changes in ionic composition as Na⁺,K⁺, Ca²⁺ and Mg²⁺, osmolality inseminal fluid, percentage of motile spermatozoaand velocity were investigated in response toCPP and different dosage of LHRHa. The lowestvelocity of sperm was observed after use CPPtreatment. The velocity of spermatozoa,significant main effect of the treatment(P < 0.0001) and the time of sperm collection(P < 0.0104) were evaluated. The osmolality ofseminal fluid was different betweenexperimental groups of LHRHa (48.0–62.7mOsmol.kg^–1) and CPP (33.0–46.3mOsmol.kg^–1) treatments. The osmolalitywas significantly higher on the first day andone-half, then declined on day three, rangingfrom 33.0 to 62.7 mOsmol.kg^–1. Analysisof variance showed significant main effects ofthe treatment (P < 0.0001) and the time ofsperm collection (P < 0.0002) on the osmolalityof seminal fluid. The level of Na⁺ andK⁺ ion was different between experimentalgroups of LHRHa and CPP treatment. The highestconcentration of 11.11 mmol.l^–1 wasobserved at Na⁺ ion. Then theconcentrations declined on the level 1.56, 0.52and 0.36 mmol.l^–1 for K⁺, Ca²⁺and Mg²⁺ ions, respectively. There werehighly positive correlations between osmolalityof seminal fluid and dosage of LHRHa treatment(r = 0.84), velocity of spermatozoa andosmolality of seminal fluid (r = 0.57) andosmolality of seminal fluid and Na⁺concentration at seminal fluid (r = 0.70).Injection with LHRHa increased quality of spermas velocity of sperm, level of Na⁺,K⁺ and osmolality at seminal fluidcompared to CPP treatments.     

Relative importance of motility and antibiosis in the rhizoplane competence of a biocontrol agent Pseudomonas fluorescens MelRC2Rif

Mutants defective in motility or antibiotics production were obtained by Tn5 mutagenesis of a biocontrol agent Pseudomonas fluorescens MelRC2Rif (wt). Tomato or melon seeds were co-inoculated with a Tn5 mutant and wt in a 1:1 ratio and then grown in soil for 10 days. There was no change in ratios of Tn5 mutants defective in antibiosis to wt in the process of rhizoplane colonization, suggesting little contribution of in vitro antibiosis to the rhizoplane competence of P. fluorescens MelRC2Rif. Similar results were also obtained when seeds treated with bacteria were planted in soil artificially infested with fungal pathogens. In contrast, ratios of Tn5 mutants defective in motility to wt significantly decreased, suggesting the contribution of motility to the rhizoplane competence of this bacterium. When a non-motile Tn5 mutant and wt were co-inoculated into soil at a matric potential of pF 2.3 (–20 kPa) and plants were then grown, there was no change in the ratio in rhizoplane colonization, suggesting that motility might have a role in the movement along roots but an insignificant role in the movement from bulk soil towards roots. When they were co-inoculated into 0.2% water agar (WA) instead of soil, a remarkable decline in ratios was detected. Thus it was soil structure that hindered the efficiency of motility. Time course enumeration of rhizoplane colonization of tomatoes grown in WA revealed that motility was an important means of movement towards and/or along roots rather than the multiplication on roots. Received: 8 July 1996     

牛精液中药复方稀释液配方筛选研究

在种公牛精液商业稀释液的基础上,按6%、12%、24%和48%的4个水平向稀释液中分别添加4种中药复方提取液,制作0.25 ml细管冻精,解冻(40 ℃,10 s)后比较分析精子活率、顶体完整率、质膜完整性3个指标,以确定较优的中药稀释液配方.结果表明:添加了中药复方提取液的稀释液能更有效的冷冻保存牛精子细胞.其中,复方一和复方二的冷冻效果最佳.复方一在添加量为12%时精子活率、顶体完整率及质膜完整率分别为41.50%、40.14%和38.16%(P＜0.05),分别比对照组提高了10.67%、3.92%和5.50%;复方二在添加量为6%时,精子活率、顶体完整率、质膜完整率3项指标分别达到41.33%、41.59%和40.00%(P＜0.05),分别比对照组提高了10.50%、5.37%和7.34%.     

细菌鞭毛系统的研究进展

鞭毛是许多细菌用于推动自身运动的细胞器。运动能够帮助细菌趋向有利环境或躲避有害环境。鞭毛还在细菌底物吸附、生物膜形成和病原侵染过程中起着很重要的作用。因此,鞭毛对细菌的生存和与其他细菌的竞争至关重要。鞭毛的表达需要超过40个基因的参与,其合成和工作是一个非常耗能的过程,所以其基因的表达和组装是受到严格调控的。对鞭毛系统的研究有助于更深入地了解基因的表达调控机理,认识微生物对环境的适应性和应用仿生学制造机械马达等。该研究将重点关注于鞭毛系统的表达调控。     

Hyperactivated Sperm Motility: Are Equine Sperm Different?

A recent study has suggested a central role for hyperactivated sperm motility in successful equine in vitro fertilization, as the highest apparent fertilization rates reported yet were obtained using exogenous stimulation of hyperactivated motility in sperm that had been incubated for capacitation. Hyperactivated motility has been studied intensively in other species, but little data are available in this area in the horse. Hyperactivated motility is associated with an influx of calcium from the extracellular space, and in other species this occurs because of an increase in intracellular pH during capacitation. Influx of calcium seems to trigger changes in flagellar function through calcium–calmodulin–calmodulin kinase-related effects on dynein function, but the exact mechanisms resulting in hyperactivated motility are still unclear. We have been exploring the physiology of hyperactivated motility in stallion spermatozoa and have found many of the mechanisms present in other species to be in place in the stallion. Further research is needed to determine why stallion spermatozoa seem to fail to undergo hyperactivation in response to capacitating environments that support this activity in other species.     

测定冻精酶活性的高低预测精液品质

[目的]为了通过测定牛冻精谷丙转氨酶(GPT)和谷草转氨酶(GOT)的活性高低来预测精液品质.[方法]用人医血清酶类测定的赖氏法测定两种酶的活性.[结果]表明:谷丙转氨酶与精子活率呈极显著负相关(P＜0.01),与顶体完整率呈显著负相关(P＜0.05);谷草转氨酶与精子活率呈极显著负相关(P＜0.05),与顶体完整率呈极显著负相关(P＜0.01).[结论 ]谷丙转氨酶和谷草转氨酶活性愈高,精子活率和顶体完整率愈低,精子品质下降.     

Systemic effects of a prolonged continuous infusion of ketamine in healthy horses

Background: Ketamine as continuous rate infusion (CRI) provides analgesia in hospitalized horses. Objective: Determine effects of prolonged CRI of ketamine on gastrointestinal transit time, fecal weight, vital parameters, gastrointestinal borborygmi, and behavior scores in healthy adult horses. Animals: Seven adult Thoroughbred or Thoroughbred cross horses, with permanently implanted gastric cannulae. Methods: Nonblinded trial. Random assignment to 1 of 2 crossover designed treatments. Ketamine (0.55 mg/kg IV over 15 minutes followed by 1.2 mg/kg/h) or lactated Ringer's solution (50 mL IV over 15 minutes followed by 0.15 mL/kg/h) treatments. Two hundred 3 × 5 mm plastic beads administered by nasogastric tube before drug administration. Every 2 hours vital parameters, behavior scores recorded, feces collected and weighed, and beads retrieved. Every 6 hours gastrointestinal borborygmi scores recorded. Study terminated upon retrieval of 180 beads (minimum 34 hours) or maximum 96 hours. Nontransit time data analyzed between hours 0 and 34. Results: No significant (P < .05) differences detected between treatments in vital signs or gastrointestinal borborygmi. Significant (P = .002) increase in behavior score during ketamine infusion (0.381) from hours 24–34 compared with placebo (0). Ketamine caused significant delay in passage of 25, 50, and 75% of beads (ketamine = 30.6 ± 5.3, 41.4 ± 8.4, 65.3 ± 13.5 hours versus placebo = 26.8 ± 7.9, 34.3 ± 11.1, 45.8 ± 19.4 hours), and significant (P < .05) decrease in fecal weight from hours 22 (12.6 ± 3.2 versus 14.5 ± 3.8 kg) through 34 (18.5 ± 3.9 versus 12.8 ± 6.4 kg) of infusion. Conclusions and Clinical Importance: Ketamine CRI delayed gastrointestinal transit time in healthy horses without effect on vital parameters.     

Reference Values and Repeatability of Transabdominal Ultrasonographic Gastrointestinal Tract Thickness and Motility in Healthy Donkeys (Equus asinus)

The present study aimed to provide reference ranges for the wall thickness and motility pattern of the gastrointestinal tract from a sample of donkeys (Equus asinus) population using B-mode ultrasonography. In the present study, 30 clinically healthy donkeys (Equus asinus) (15 males and 15 females), aged 2–20 year old and weighed 100–280 kg were randomly selected for B-mode ultrasonographic scanning of the abdomen. The wall thickness of the stomach, duodenum, jejunum, left colon, right colon, and cecum was assessed. Moreover, the motility pattern of the duodenum, jejunum, left colon, right colon, and cecum was evaluated over a period of 3 minutes. Abdominal ultrasonographic scanning of the gastrointestinal tract of healthy donkeys explored that the stomach, duodenum, jejunum, left colon, right colon, and cecum could be visualized easily. The wall thickness of the stomach, duodenum, jejunum, left colon, right colon, and cecum was 7.0 ± 0.9 mm, 3.3 ± 1.0 mm, 5.4 ± 0.6 mm, 5.1 ± 0.5 mm, 5.4 ± 0.5 mm, and 5.4 ± 0.6 mm, respectively. The thickest part of the gastrointestinal tract is the stomach, whereas the thinnest part is the duodenum. The motility pattern of the duodenum, jejunum, left colon, right colon, and cecum was 7.7 ± 1.3 contractions/3 minutes, 6.9 ± 1.1 contractions/3 minutes, 4.1 ± 1.2 contractions/3 minutes, 5.5 ± 1.3 contractions/3 minutes, and 4.0 ± 0.8 contractions/3 minutes, respectively. Both the duodenum and jejunum contractions were significantly higher than that of the left colon, right colon, and cecum. This is the first study reporting the reference values for both the wall thickness and motility pattern of the gastrointestinal tract in healthy donkeys (Equus asinus) in Egypt. Good knowledge of these standard and reference values of the wall thickness and motility pattern of gastrointestinal tract structures represents a step in the early diagnosis of the gastrointestinal disorders, including colic in such animal species.