Visualization of capsule formation by Erwinia amylovora and assays to determine amylovoran synthesis

Exopolysaccharide (EPS) synthesis by Erwinia amylovora depends on environmental and genetic predispositions. To measure the amount of the acidic EPS amylovoran synthesized by E. amylovora cell cultures, a turbidity assay using cetylpyridinium salt was developed. The EPS produced by bacteria grown on solid media was additionally characterized by its water content. The amylovoran capsules were visualized in situ by staining with fluorescein isothiocyanate (FITC)-labelled lectin from Abrus precatorius, which reacts with the galactose residue of the EPS side chain. The staining and the turbidity assays were applied to suspension cell cultures or to cells from colonies and did not require any purification steps. Lectin staining was superior to electron microscopic (EM) techniques for visualization of capsules. For EM, the capsule was stabilized with polycationic ferritin. In contrast to lectin staining, only a small fraction of the cells was found to be EPS-coated in the EM assay. An increase in capsulation and in amylovoran production was found in conjunction with mutations in a ribosomal protein conferring resistance to streptomycin. Furthermore, the presence of sorbitol in the growth environment resulted in high synthesis of amylovoran. Cells in the stationary growth phase continued to produce amylovoran. Apparently, the strong dependence of the fireblight pathogen on capsules requires the capacity for EPS synthesis in all growth stages in order to escape plant defence reactions.     

Cloning and sequencing of a gene involved in the synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 3

A 4.5 kb ScaI chromosomal DNA fragment of a clinical isolate of Streptococcus pneumoniae serotype 3 was cloned in Escherichia coli. Combined genetic and molecular analyses have allowed the localization, in a 781 by EcoRV subfragment, of a gene (cap3-1) directly responsible for the transformation of an unencapsulated, serotype 3 mutant to the capsulated phenotype. Comparison of the deduced amino acid sequence of CAP3-1 with the protein sequences compiled in the data banks revealed that the CAP3-1 polypeptide was highly similar to the amino-terminus of the GDP-mannose dehydrogenase of Pseudomonas aeruginosa, an enzyme that participates in the synthesis of the mucoid polysaccharide of this species. In addition, the 32 N-terminal amino acids of CAP3-1 perfectly matched structures common to NAD⁺-binding domains of many dehydrogenases. Our results indicate that the 4.5 kb ScaI fragment might also contain genes common to 13 different pneumococcal serogroups or scrotypes tested. To the best of our knowledge, this is the first time that a gene of the capsular complex of S. pneumoniae has been cloned and sequenced. The findings reported here provide new insights for the study of the molecular biology of the main virulence factor responsible for the pathogenesis of pneumococcal infections and might represent a basic step in the identification of cross-reactive antigens that should allow the preparation of new and improved vaccines.     

龙珍胶囊药理试验中血清谷丙转氨酶、尿素氮、肌酐的检测

本实验研究旨在考查长期服用 DTC 对大白鼠肝肾功能的影响。结果表明用 DTC 饲养3至6个月大白鼠血中,其转氨酶活力,尿素氮、肌酐的水平没有明显变化.实验结果提示 DTC 是一种安全药物,长期服用对肝肾功能的影响极微.     

Conversion of codeine to morphine in isolated capsules of Papaver somniferum

The biotransformation of codeine to morphine was studied in isolated capsules of Papaver somniferum. Cofactors such as nicotinamide adenine dinucleotide, adenosine 5′-triphosphate, S-acetyl coenzyme A and pyridoxal phosphate were not required in the conversion of codeine to morphine. Reducing agents such as dithiothreitol, glutathione and β-mercaptoethanol strongly promoted codeine and morphine degradation, while morphine formation remained at a constant level. Hydrogen peroxide (concentration > 0.25 mM) caused the conversion of codeine and morphine to N-oxides by non-enzymatic oxidation. Isolated capsules of P. somniferum provide a method of studying the biotransformation of codeine to morphine.     

First records of advanced embryos and egg capsules of Bathyraja skates from the deep north-eastern Atlantic

Five variously developed embryos [142–279 mm total length (L_T)] from egg capsules from the Porcupine Seabight, north-eastern Atlantic (1541 m depth) are used to establish the ontogeny and early life history of Bathyraja richardsoni. The capsules of this species and two half-formed ones from the shell glands of a large (1620 mm L_T) female Bathyraja pallida taken off Ireland (c. 1900 m depth) are described and illustrated. The varying degree of yolk sac absorption found in the B. richardsoni embryos is discussed in relation to hatching size and its seeming size independence over c. 20–50 mm within the embryos' total length range. The conservative variation in external morphology with development from advanced embryos to adults among such deep stenobathic Bathyraja skates is commented upon, as is the bathymetric segregation of adults from levels in which egg capsules are deposited and young develop.     

Parental size and environmental conditions affect egg capsule production by Nassarius reticulatus (Linnaeus 1758) (Gastropoda: Nassariidae)

In shallow coastal habitats scavenging netted whelks Nassarius reticulatus attached egg capsules to the stipes of red algae Chondrus crispus and occasionally on Furcellaria lumbricalis and Plumaria plumose. In the laboratory egg capsules were laid on aquaria sides and lids by individuals ≥ 21 mm shell length. Larger size classes produced more egg capsules and spawned over a longer period and in doing so partitioned less energy into shell growth. Large netted whelks (25-28.9 mm) produced larger capsules which contained significantly more and larger eggs than those produced by smaller individuals (21-24.9 mm). Egg capsule production continued throughout the year by regularly fed N. reticulatus held at ambient seawater temperatures. Egg production increased in the spring and summer with peak production during June (15 °C), decreased between August and October and resumed again during the winter (November to February at ∼ 7 °C). During the summer (15-16 °C) egg capsules were smaller and contained smaller eggs than those deposited during the winter (7-10 °C), although the number of eggs · capsule⁻¹ was similar. Enforced food limitation reduced the number and size of the egg capsules, the number and size of eggs produced · female⁻¹ and the duration of the breeding period. Hatching success of N. reticulatus egg capsules was high (95%) even at winter seawater temperatures (11-8.5 °C) and the duration of embryonic development was fastest between 15 and 17.5 °C.     

Comparison of the antinociceptive effect of celecoxib, diclofenac and resveratrol in the formalin test

The peripheral antinociceptive effect of the selective COX-2 inhibitor celecoxib in the formalin-induced inflammatory pain was compared with that of resveratrol (COX-1 inhibitor) and diclofenac (non-selective COX inhibitor). Rats received local pretreatment with saline, celecoxib, diclofenac or resveratrol followed by 50 microl of either 1% or 5% formalin. Peripheral administration of celecoxib did not produce antinociception at either formalin concentration. In contrast, diclofenac and resveratrol produced a dose-dependent antinociceptive effect in the second phase of both 1% and 5% formalin test. The peripheral antinociception produced by diclofenac or resveratrol was due to a local action, as drug administration in the contralateral paw was ineffective. Results indicate that the selective COX-2 inhibitor celecoxib does not produce peripheral antinociception in formalin-induced inflammatory pain. In contrast, selective COX-1 and non-selective COX inhibitors (resveratrol and diclofenac, respectively) are effective drugs in this model of pain.     

Simultaneous determination of celecoxib,hydroxycelecoxib, and carboxycelecoxib in human plasma using gradient reversed-phase liquid chromatography with ultraviolet absorbance detection

A new HPLC method for the simultaneous determination of celecoxib, carboxycelecoxib and hydroxycelecoxib in human plasma samples has been developed. Following a solid-phase extraction procedure, the samples were separated by gradient reversed-phase HLPC (C(18)) and quantified using UV detection at 254 nm. The method was linear over the concentration range 10-500 ng/ml. The intra-assay variability for the three analytes ranged from 4.0 to 12.6% and the inter-assay variability from 4.9 to 14.2%. The achieved limits of quantitation (LOQ) of 10 ng/ml for each analyte allowed the determination of the pharmacokinetic parameters of the analytes after administration of 100 mg celecoxib.     

Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells

Although the influence of selective cyclooxygenase (COX)-2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX-2 inhibitors. Celecoxib strongly suppressed the proliferation of COX-2 expressing HT-29 cells at 10-40 microM. NS-398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT-29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase-3/7 activation. DNA fragmentation was also increasd in COX-2 non-expressing cell lines (SW-480 and HCT-116) by exposure to celecoxib for 6-24 h. All six COX-2 inhibitors suppressed the production of prostaglandin E(2) by HT-29 cells, suggesting that the pro-apoptotic effect of celecoxib was unrelated to inhibition of COX-2. Inactivation of Akt might explain the differential pro-apoptotic effect of these selective COX-2 inhibitors on colon adenocarcinoma cells.     

Prevention by celecoxib of secondary hyperalgesia induced by formalin in rats

Administration of formalin in rat paws results in stimulation of nociceptive pathways, which leads to an increase in the excitability of neurons present in dorsal horn. This increased neuron excitability, described as central sensitization, may result in development of inflammatory pain at a distant site of injury application, known as secondary hyperalgesia. The aim of the present study was to verify whether formalin injection in rat paws would lead to secondary hyperalgesia development, as measured by the tail-flick test. We also aimed to investigate whether celecoxib, a specific cyclooxygenase 2 (COX-2) inhibitor, would affect secondary hyperalgesia. Formalin injected into the rat paws significantly reduced the latency for a flick response in the rat tail, which characterized development of secondary hyperalgesia. In addition, formalin-induced secondary hyperalgesia was locally prevented by pre-but not post-celecoxib treatment. However, celecoxib administered spinally inhibited formalin-induced secondary hyperalgesia, either administered previously or following formalin. In contrast, piroxicam, an unspecific COX inhibitor which displays an increased selectivity towards COX-1, only prevented secondary hyperalgesia to formalin at a high dose following spinal administration. Taken together, these results suggest that COX-2 plays an important role both in the central and in the peripheral nerve sensitization following formalin administration in rat paws. They also suggested that once central sensitization starts it can no longer be blocked by a specific COX-2 inhibitor administered locally. Notwithstanding, spinal administration of a specific COX-2 inhibitor still blocks ongoing sensitization and prevents maintenance of central sensitization.