NMR assignment of the nonstructural protein nsp3(1066–1181) from SARS-CoV

Sequence-specific NMR assignments of the globular core comprising the residues 1066–1181 within the non-structural protein nsp3e from the SARS coronavirus have been obtained using triple-resonance NMR experiments with the uniformly [¹³C, ¹⁵N]-labeled protein. The backbone and side chain assignments are nearly complete, providing the basis for the ongoing NMR structure determination. A preliminary identification of regular secondary structures has been derived from the ¹³C chemical shifts.     

Elimination ofMycoplasma hyorhinis infections from four cell lines

Summary Four monolayer mammalian cell lines were cured ofMycoplasma hyorhinis infections by cloning in microtiter dishes in the presence of tetracycline and kanamycin. During cloning, cultures were refed with fresh antibiotic containing medium every 2 or 3 d for 14 d and were then cultured without effective antibiotics for at least 21 d. From the four lines we recovered 29 clones, none of which were infected after treatment as judged by the lack of extranuclear fluorescence after staining with the fluorochrome Hoechst 33258, and by normal autoradiographic labeling of the cells by tritiated nucleosides. One clone from each line was tested further by attempted culture of mycoplasmas and was also judged to be uninfected. Infection has not reappeared in any of the clones after extensive culture in the absence of the effective antibiotics. This investigation was supported by Public Health Service Research Grant GM26137 from the National Institutes of General Medical Sciences, National Institutes of Health, Bethesda, MD.     

Inoculation and acquisition of maize bushy stunt mycoplasma by its leafhopper vector Dalbulus maidis

Maize bushy stunt mycoplasma (MBSM), a mycoplasma-like organism, is transmitted in a persistent manner by the corn leafhopper, Dalbulus maidis, to maize (Zea mays). The influence of the duration of acquisition access and inoculation access periods on the transmission of MBSM by D. maidis was investigated. The proportion of plants infected by D. maidis increased significantly from 0 to 0.51 as the inoculation access time to a plant increased from 10 min to 72 h (X²= 101.5, P < 0.001). Likewise, the proportion of insects acquiring MBSM from infected plants increased from 0 to 0.19 as the acquisition access time to the source plant increased from 10 min to 72 h (X²= 53.2, P < 0.001). The data were fitted to a loglinear regression model. No significant association was found between the sex of the insects and vector ability.     

Glial Alkalinization Detected In Vivo by 1H-15N Heteronuclear Multiple-Quantum Coherence-Transfer NMR in Severely Hyperammonemic Rat

Abstract: Brain [5-¹⁵N]glutamine amide protons were selectively observed in vivo by ¹H-¹⁵N heteronuclear multiple-quantum coherence-transfer NMR in spontaneously breathing, severely hyperammonemic rats during intravenous [¹⁵N]ammonium acetate infusion and the subsequent recovery period. The linewidth of brain [5-¹⁵N]-glutamine amide proton H_z increased from 36 ± 2 Hz at 3.4 h to 58 ± 6 Hz after 5.7 h of infusion, a net increase of 22 ± 6 Hz. Concomitantly, brain ammonia concentration increased from 1.7 to 3.5 ± 0.2 µmol/g and the rat progressed from grade III to grade IV encephalopathy. On recovery to grade III and decrease of brain ammonia concentration to 1.3 µmol/g, the linewidth returned to 37 ± 2 Hz. In aqueous solution, [5-¹⁵N]glutamine amide proton H_z underwent a 17-Hz linebroadening when pH was raised from 7.1 to 7.5 at 37°C, due to the increased rate of base-catalyzed exchange with water proton. Hence, linebroadening is a sensitive measure of changing intracellular pH. The 22-Hz linebroadening observed in vivo in severely hyperammonemic grade IV rats strongly suggests that the intracellular pH increases from 7.1 to about 7.4–7.5 in astrocytes where glutamine is synthesized and mainly stored. Probable mechanisms for the ammonia-induced alkalinization and decreased intraglial buffering capacity, as well as implications of the result for pathogenesis of hepatic encephalopathy, are discussed.     

Mycoplasma detection by PCR analysis

Summary The polymerase chain reaction (PCR) method was used to detect mycoplasma contamination in a panel of 42 continuous cell lines. According to the microbiological cultivation assay on agar, 29 cell lines were chronically infected and 13 cell lines were negative. Sets of outer and inner primers (nested double-step PCR) were applied which anneal to DNA sequences coding for conserved regions of the 16S rRNA. These oligonucleotides allow for the amplification of DNA regions found in at least 25 mycoplasma species (including the ones most commonly found in cell cultures), but do not cross-hybridize with DNA from eukaryotic cells. Mycoplasma-positive cell lines showed distinctive bands in ethidium bromide-stained gels, both after the first round of amplification as well as after the second PCR; all agar-negative cell lines were also unambiguously negative in the PCR assay. Thus, neither false-positive nor false-negative results occurred. Provided that the proper PCR working conditions are scrupulously observed, the PCR amplification has several outstanding advantages: high sensitivity, specificity, reliability, objectivity, speed, and simplicity.     

MORBILLIVIRUS INFECTION IN TWO SPECIES OF PILOT WHALE (GLOBICEPHALA SP.) FROM THE WESTERN ATLANTIC

We report evidence of enzootic morbillivirus infection among long-finned, Globicephala melas, and short-finned, G. macrorhynchus, pilot whales in the western Atlantic. A retrospective serologic survey, using five morbilliviruses, was carried out on 99 G. melas from 14 stranding events between 1982 and 1993 and from 25 G. macrorhynchus stranded in 5 events between 1986 and 1994. A blood sample was also obtained from an adult G. melas by-caught in the western North Atlantic. Tissues were collected from 24 G. melas and 15 G. macrorhynchus for histology and immunoperoxidase staining. Neutralizing antibody titers were found in 92 (92%) of 100 G. melas and 16 (64%) of 25 G. macrorynchus, and titers were highest against cetacean morbilliviruses. Seroprevalence was similar between age classes and sexes. The earliest evidence of infection was in a G. melas that stranded in 1982. Stable antibody titers were observed in pilot whales under rehabilitation for up to eight months. Clinical disease consistent with morbillivirus pneumonia was detected in a G. melas calf. Immunoperoxidase staining confirmed that viral antigen was present in the lesions. We propose that enzootic infection in pilot whales is facilitated by population size, social structure, and migration patterns. Furthermore, through mixing with other odontocetes, pilot whales could act as vectors through the Atlantic. Clinical morbillivirus infection may precipitate mass strandings of highly social odontocetes.     

呼吸机相关性肺炎患者病原菌分布及IL 35的炎症控制机制

目的探讨呼吸机相关性肺炎(VAP)患者病原菌分布及白细胞介素35(IL-35)的炎症控制机制。方法回顾性分析2016年12月至2018年12月在我院重症监护病房(ICU)行机械通气的152例患者的临床资料,根据是否发生VAP将其分为VAP组(n=70)和对照组(n=82)。收集VAP患者痰液进行细菌培养及药敏实验,检测并比较两组患者外周血单个核细胞(PBMCs)中CD4~+CD25~+CD127dim/-调节性T细胞(Tregs)比例及血清中白细胞介素35(IL-35)、肿瘤坏死因子α(TNF-α)、干扰素-γ(IFN-γ)水平;同时检测并比较经佛波酯和重组人IL-35刺激后的Tregs细胞培养液上清中IL-35、IFN-γ、TNF-α水平。结果 70例VAP患者共检出病原菌128株,其中革兰阴性菌占82.81%,革兰阳性菌占14.84%,真菌占2.34%。鲍曼不动杆菌对头孢西丁的耐药率最高,对头孢哌酮的耐药率最低。铜绿假单胞菌对头孢西丁的耐药率最高,对亚胺培南的耐药率最低。肺炎克雷伯菌对头孢西丁的耐药率最高,对头孢哌酮的耐药率最低。VAP组患者PBMCs中CD4~+CD25~+CD127dim/-Tregs比例及血清中IL-35、IFN-γ、TNF-α水平显著高于对照组(t=12.675、36.631、13.464、8.482,均P0.001)。经重组人IL-35刺激后的Tregs细胞培养液上清中IL-35水平显著高于佛波酯刺激后(t=8.886,P0.001)。经重组人IL-35刺激后的Tregs细胞培养液上清中IFN-γ、TNF-α水平显著低于佛波酯刺激后(t=14.091、7.588、均P0.001)。结论呼吸机相关性肺炎病原菌以革兰阴性菌为主,呈现多药耐药性,临床应合理使用抗菌药物以防治呼吸机相关性肺炎。IL-35可通过调控Tregs功能降低促炎因子分泌从而在呼吸机相关性肺炎发病过程中发挥抗炎症作用,提示IL-35可能为呼吸机相关性肺炎的治疗提供新的方向。     

Mycoplasma pneumoniae infection of intact guinea pig tracheas cultured in a unique matrix-embed/perfusion system

Summary A new method for the in vitro culture of entire, intact tracheas from adult guinea pigs is described. Matrix-embed/perfusion (MEP) culture is based on an immobilization of the tissue in nutrient agar. The tubular piece of agar-embedded organ was contained in a special perfusion block with two wells for liquid medium at either end. When incubated on a rocker platform, liquid medium flows through the trachea and supplies oxygen and nutrients. In this configuration, tracheas maintain near-normal metabolism (ATP content and dehydrogenase activity), structure (as determined by light and electron microscopy), and function (ciliary motion). Tissues could be maintained in vitro in a normal state for at least 4 wk, with reduced ciliary motion and cell metabolism detectable for at least 6 wk. Agarembedded tissues from the MEP cultures were nearly identical to those cultivated with standard tracheal ring explant techniques. Tracheas in the MEP cultures were infected withMycoplasma pneumoniae. Attachment was neuraminidase-sensitive. Mycoplasma attachment was lowest on the epithelium along the dorsal ridge, but was uniform along the length of the trachea. Ciliostasis and cytonecrosis induced byM. pneumoniae was dose dependent. The matrix-embed/perfuse technique appears to have considerable potential for several types of in vitro studies on trachea or other tubular organs. This study was supported in part by USPHS Grants AI 12559 and AI 17795 from the National Institutes of Allergy and Infectious Diseases, and Grants HL 23806 and HL 26880 from the National Heart, Lung, and Blood Institute.     

Mycoplasma detection in cell culture by concomitant use of bisbenzamide and fluoresceinated antibody

Summary Conditions are presented for application of both bisbenzamide (Hoechst 33258) stain and a specific fluoresceinated anti-Mycoplasma hyorhinis IgG to a single cell culture preparation. This allows the same field on a slide to be viewed for presumptive diagnosis of any cell culture contaminant mycoplasma by bisbenzamide staining and for definitive diagnosis ofM. hyorhinis strains using fluoresceinated antibody. The use of this method plus a cultural procedure will permit identification of the “noncultivable”M. hyorhinis strain DBS 1050.