Biomedical subjects
Publications and source records attributed to F Qu.
Amperometric detection in liquid chromatography is introduced in this article. Detection mechanism and conditions involved in the application in HPLC and HPIC with d.c. amperometry, pulsed amperometry and integrated amperometry detection are discussed.
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Previous studies on turnip crinkle virus (TCV) have suggested that the two small, centrally located ORFs, conserved in all Carmoviruses, are both required for cell-to-cell movement (Hacker et al., 1992). We now demonstrate that the cell-to-cell movement of TCV is mediated by in trans complementation of the two proteins. First, both of the putative movement proteins (MPs p8 and p9) were shown to be translated in vitro from transcripts representing the 1.7-kb subgenomic RNA. Western blot analysis, using antisera prepared against GST fusion proteins of both genes, was then used to show that the p8 but not the p9 protein accumulated to detectable levels in particulate fractions of infected cells. Cell-to-cell movement of various MP mutants in Arabidopsis was evaluated by in situ hybridization of inoculated leaves. Changes in either of the two MP genes resulted in failure of the mutants to move cell-to-cell. Coat protein was found to be unnecessary for cell-to-cell movement. Complementation of cell-to-cell movement by co-inoculating p8-defective mutants with a p9-defective mutant resulted in delayed systemic infection. In contrast, efficient cell-to-cell movement was achieved when the MP mutants were inoculated into transgenic plants expressing the corresponding functional gene(s). These experiments provide further evidence that both MP genes encoded by Carmoviruses must function in trans in the same cell in order to mediate cell-to-cell movement.
A polymerase chain reaction assay based on differences in the internal transcribed spacer regions of ribosomal DNA was developed for distinguishing 2 members of the Anopheles dirus sibling species complex. This assay distinguished An. dirus species A from species D by producing diagnostic bands, 374 base pairs (bp) in length for species A and 663 bp in length for species D. Both laboratory colonies and field collections from Hainan and Yunnan provinces of China were identified with 100% accuracy.
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AIM: To distinguish cryptic species A and D of Anopheles dirus complex using polymerase chain reaction (PCR). METHODS: A diagnostic PCR assay of species was developed by use of three primers, one derived from highly conservative 5.8 S coding sequences and two from different interspecies sequence in the second internal transcribed spacer (ITS2) of ribosomal DNA. RESULTS: Using the PCR method, specific fragments were amplified in both species, the size of fragments is 374 bp for species A and 663 bp for species D. Thirty samples of species A from AFRIMS and HN laboratory colony and seven samples of the species D from Yunnan Province were correctly identified by PCR. Satisfactory results were obtained from the amount of DNA as little as 1/1,600 of extracted DNA of a single mosquito or 1/5 of DNA derived from one leg of a mosquito triturated in water. A total of 148 field-collected specimens of Anopheles dirus from Heping(HP), Baisha(BS), Loukui(LK), and Maoyang (MY) in Hainan Province revealed fragment characteristic of species A, while 30 specimens from Mengla (ML) in Yunnan Province showed the specific fragment of species D. CONCLUSION: A simple and reliable method was developed to identify cryptic species A and D of Anopheles dirus complex and it was further verified that Anopheles dirus from Hainan and Yunnan Provinces is the species A and the species D, respectively.
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A protoplast infection assay has been used to reliably examine the viral RNA encapsidation of turnip crinkle virus (TCV). Analysis of the encapsidation of various mutant viral RNAs revealed that a 186-nucleotide (nt) region at the 3' end of the coat protein (CP) gene, with a bulged hairpin loop of 28 nt as its most essential element, was indispensable for TCV RNA encapsidation. When RNA fragments containing the 186-nt region were used to replace the CP gene of a different virus, tomato bushy stunt virus, the resulting chimeric viral RNAs were encapsidated into TCV virions. Furthermore, analysis of the encapsidated chimeric RNA species established that the RNA size was an important determinant of the TCV assembly process.
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A rice U3 small nuclear RNA (snRNA)-encoding gene has been isolated. The coding region of this gene contains all five conserved sequence boxes common to plant U3 snRNAs. The upstream and downstream regions of the gene harbour characteristic sequence elements required for transcription with RNA polymerase III (pol III), as well as three monocot-specific promoter (MSP) elements [Connelly et al., Mol. Cell Biol.14 (1994) 5910-5919], two of them comprising a palindromic G+C-rich segment. The sequence (TTTAAAA) of the TATA-box in this gene does not fit the established consensus [Marshallsay et al., Plant Mol. Biol. 19(1992) 973-983], making this gene unique among reported snRNA-encoding genes of plants. An RNase protection assay showed that the gene is expressed properly in cucumber protoplasts. We thus suggest that, with other promoter elements present, the TATA-box for RNA pol III-specific snRNA-encoding genes may not be as well conserved as that for RNA pol II-specific genes.
After the unexpected appearance of lethal symptoms on tomato plants infected with the PSTVd strain Intermediate Di, viroids were isolated and sequenced. It was found that a new strain, named RG 1, had been generated spontaneously in our greenhouse. In a different series of plant passages two new strains, named QF A and QF B, were detected which coexisted with the wild-type strain Di. Strains QF A and QF B showed intermediate symptoms when inoculated separately. In order to confirm the working hypothesis that the more pathogenic strain outcompetes the less pathogenic strain but strains of similar pathogenicity might coexist in the host, strains of different pathogenicity were mixed for inoculation in a ratio from 1:1 to 1:100 (more pathogenic:less pathogenic). The concentrations of the individual strains were determined 6 weeks postinfection with the method of nondenaturing polyacrylamide gel electrophoresis, and the working hypothesis was confirmed. The total concentrations of viroids in infected plants were very similar, irrespective of whether severe, intermediate, or mild strains or mixtures of different strains were present. The mutations in all new strains (3 in RG 1, 2 in QF A, 3 in QF B) were located in the so-called virulence-modulating region. The mutations of strain RG 1 influenced dramatically the thermodynamic stability of the native rod-like structure, as determined experimentally by temperature-gradient gel electrophoresis. Since during replication a multihairpin structure is generated transiently which is transformed afterwards into the rod-like structure, a lower thermodynamic stability of the rod-like structure leads to a higher accumulation of the transient structure. It is assumed that the transient structure, which is active in replication as shown earlier, is essential also in pathogenesis. This model explains the experimentally determined interdependence between pathogenicity and replicability of PSTVd strains.
Five clones of monoclonal antibodies (McAbs) were generated from BALB/c mice which were activized with PPD and immunized with esterases extracted from malathion-resistant Culex quinquefasciatus. All 5 clones of McAbs belonged to the IgG1 subtype, additive index (A.I.) of every two McAbs did not exceeded 50%. After a 1 h of incubation, the McAbs at the titer of 1:1,000 completely inhibited the activity of the amplified esterases. Of 8 strains of adult mosquitoes studied by Western-blot assay, all exhibited the 64 kDa esterase band recognized by the McAbs, six (SP, FS, SH, CD, RD, RM) showed more bands of different MW (43-60 kDa), the higher the organophosphorus resistant level, the more and the denser the bands. The above McAbs had little or no effect against the esterases of dimethrin-resistant strain and the larva stage of malathion-resistant Culex quinquefasciatus.
Neurohypophysial arginine vasopressin (AVP) concentrations were determined by RIA in 9 patients with cerebral infarction, 11 patients with intracerebral hemorrhage, 5 patients with subarachnoid hemorrhage and 5 control subjects. The results showed that neurohypophysial AVP concentration in patients with cerebral infarction increased by 225.8% as compared with the control subjects (P < 0.05), and the AVP concentrations in patients with intracerebral and subarachnoid hemorrhage did not change significantly. Brain herniation as a result of intracranial hypertension, gastrointestinal bleeding and hyperglycemia (two clinical manifestations of stress) was seen more frequently in hemorrhagic stroke patients than in ischemic stroke patients. These findings suggest that the ischemic brain damage may contribute to the elevation of neurohypophysial AVP concentration in patients with cerebral infarction.
Different strains of Culex mosquitoes (Cx. pipiens quinquefasciatus and Cx. Pipiens pallens) were immunologically-detected for organophosphate resistance with anti-esterase monoclonal antibody, and the results were compared with those detected by bioassay and biochemical microplate assay. It was found that the resistance-detection-rate detected by immunologic methods were higher than the corresponding data detected by the biochemical method, and the levels of resistance detected by sandwich-ELISA were higher than the corresponding levels detected by bioassay and the biochemical method. The thresholds for resistance in sandwich-ELISA were (at absorbence 450) > or = 0.5, and in microplate assay were (at absorbence 590) > or = 2.5 x 10(-3) mumol/min.mg protein. Dot-ELISA method was developed to meet the requirement of the field test and proved to be fast and convenient, especially in the detection of samples with higher esterase activity.
Auditory brainstem response and auditory cortex response were recorded repeatedly in 35 guinea pigs after exposure to intensive white noise (125 dB, 150 min.) for 62 d. the amplitude of evoked potential of acoustic nerve was decreased by 29% (P < 0.05), of the cochlear nuclei by 28% (P < 0.05). However, the amplitude of response of superior olives nuclei was increased by 21% (P < 0.05), of the inferior colliculi by 37% (P < 0.05), of the cortical evoked response by 131% (P < 0.001). The results indicate that the amplitudes of auditory evoked potential showed a centripital augmentation after exposure to intensive noise. The centripital augmentation was observed not only during the period of the temporary threshold shift (TTS) but also during that of the permanent threshold shift (PTS).
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From site-directed mutagenesis of potato spindle tuber viroid (PSTVd) it had been concluded earlier that the formation of a thermodynamically metastable structure containing hairpin II (HP II) is critical for infectivity. In order to differentiate between structural and sequence effects, in the present work base pairs in HP II were exchanged by site-directed double mutations without significant alterations in the native rod-like structure of PSTVd. The mutants were viable and genetically stable in the first generation, but one of the two mutations reverted to the wild-type nucleotide in the second generation. Single-site mutations in the stem of HP II, which had been described as revertants to the wild-type sequence earlier, were analysed with respect to the time course of reversion and the sequence variation during reversion. All replicating sequence variants were separated by gel electrophoretic techniques and the sequences and their relative frequencies were determined. From both types of studies it can be concluded (i) that HP II is a functional element in the (-)strand replication intermediate, generated due to sequential folding during synthesis, and that it is essential for template activity of (+)strand synthesis; (ii) that G:U pairs are tolerated transiently in (-)strand HP II; the lower stability of such a HP II is compensated by additional mutations outside HP II which suppress the competition of a rod-like structure; and (iii) that the reversions are generated spontaneously during (-)strand synthesis. Furthermore, the double-stranded structure of HP II is the essential element for short term replication of PSTVd but the exact sequence of the wild-type proves to be superior with regard to fitness and replicability of PSTVd.
Changes in the somatostatin-like immunoreactivity (SLI) concentrations in gastric antral mucosa were detected by RIA following microinjections of synthetic somatostatin (SS) or cysteamine (CSH) into the ventromedial nucleus of the hypothalamus (VMH). SLI concentrations in the antral mucosa were increased by 60.8% (p less than 0.001) and 42.3% (p less than 0.05), respectively, one and four hours after microinjection of somatostatin (0.5 microgram/0.5 microliter) into the VMH, and decreased by 32.6% (p less than 0.05) four hours after microinjection of cysteamine (15 microgram/0.5 microliter) into the VMH. Moreover, microinjection of somatostatin (0.5 microgram/0.5 microliter) into the VMH could only elicit an increase of 16.0% (p less than 0.05) in the SLI concentration in the antral mucosa one hour after bilateral subdiaphragmatic vagotomies, but still produced an increase of 120.0% (p less than 0.05) following celiac ganglionectomies. In conclusion, somatostatin (exogenous and endogenous) in the VMH seems to affect the gastric somatostatin levels via the vagal nerves.