An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus

A new SARS animal model was established by inoculating SARS coronavirus (SARS-CoV) into rhesus macaques (Macaca mulatta) through the nasal cavity. Pathological pulmonary changes were successively detected on days 5-60 after virus inoculation. All eight animals showed a transient fever 2-3 days after inoculation. Immunological, molecular biological, and pathological studies support the establishment of this SARS animal model. Firstly, SARS-CoV-specific IgGs were detected in the sera of macaques from 11 to 60 days after inoculation. Secondly, SARS-CoV RNA could be detected in pharyngeal swab samples using nested RT-PCR in all infected animals from 5 days after virus inoculation. Finally, histopathological changes of interstitial pneumonia were found in the lungs during the 60 days after viral inoculation: these changes were less marked at later time points, indicating that an active healing process together with resolution of an acute inflammatory response was taking place in these animals. This animal model should provide insight into the mechanisms of SARS-CoV-related pulmonary disease and greatly facilitate the development of vaccines and therapeutics against SARS.     

Association of the Pro12Ala and C1431T polymorphism of the PPAR gamma2 gene and their haplotypes with obesity and type 2 diabetes

OBJECTIVE: To study the association of the Pro12Ala and C1431T polymorphism of the PPAR gamma2 gene and their haplotypes with obesity and type 2 diabetes in Chinese population. METHODS: PCR-restriction fragment length polymorphism was used to determine the Pro12Ala and C1431T polymorphisms in 207 patients with type 2 diabetes and 101 non-diabetic control subjects. RESULTS: (1) In non-diabetic control population, the Ala allele frequency was 0.064, the T1431 allele frequency was 0.252. Haplotype analysis showed that the Pro12Ala and C1431T polymorphisms were in linkage disequilibrium (Do=0.63, r(2)=0.074), which constituted three major haplotypes Pro-C, Pro-T and Ala-T. (2) There were no significant differences of the distribution frequencies of the Pro12Ala and C1431T polymorphism and their haplotypes between the type 2 diabetes mellitus group and non-diabetic control group (P > 0.05). (3) The Pro12Ala polymorphism was associated with blood pressure and lipidemia in diabetic patients. The Ala allele significantly decreased the diastolic blood pressure of non-obese diabetic patients (P < 0.05), but it did not benefit to the obese diabetic patients for the lipidemia (P < 0.05). The C1431T polymorphism was associated with overweight and obesity in diabetic patients. The T1431 allele frequency in the body mass index >/= 25 layer was significantly higher than that in the body mass index < 25 layer (P < 0.05). CONCLUSION: The Pro12Ala and C1431T polymorphisms of the PPAR gamma2 gene might not be a major etiological factor for type 2 diabetes; the C1431T polymorphism was associated with overweight or obesity in diabetic patients.     

抗人CD25分子单链抗体基因的构建、表达及初步鉴定

目的:构建和表达抗人CD25分子单链抗体(scFv)蛋白,并测定其生物学活性。方法:用RT-PCR方法从能分泌特异性抗CD25单克隆抗体(mAb)的杂交瘤细胞中分离纯化抗体VH和VL基因。用重叠延伸PCR方法将VH和VL拼接在一起,构建抗CD25分子scFv的基因。将scFv基因克隆至pMD18T,用限制性内切酶切以及测序鉴定。将scFv基因连接到pBAD/gⅢA表达载体,转化Top10表达菌。阳性克隆用左旋阿拉伯糖诱导4 h,SDS-PAGE电泳检测蛋白纯度,竞争抑制ELISA实验检测其活性。结果:scFv基因长度约为700 bp。通过DNA序列测定和分析,构建出VL-(GGGGS)3-VH(但其中349位G突变为A,使Linker其中一位Gly→Ser)。其VH隶属于小鼠Ig重链可变区Ⅲ(C)亚类,全长351 bp,可编码117个氨基酸;其VL隶属于小鼠Igκ轻链可变区Ⅳ亚类,全长318 bp,可编码106个氨基酸。TOP10中表达的scFv抗体加上同时融合表达的两个标签6×His和C-mycMr约为31 000,结果符合scFv的Mr。竞争抑制细胞ELISA实验显示表达的scFv具有活性。结论:此scFv基因的表达产物具有一定的特异结合活性。为抗CD25 scFv的临床应用打下了基础。     

Origin and primary dispersal of the Mycobacterium tuberculosis Beijing genotype: clues from human phylogeography

We suggest that the evolution of the population structure of microbial pathogens is influenced by that of modern humans. Consequently, the timing of hallmark changes in bacterial genomes within the last 100,000 yr may be attempted by comparison with relevant human migrations. Here, we used a lineage within Mycobacterium tuberculosis, a Beijing genotype, as a model and compared its phylogeography with human demography and Y chromosome-based phylogeography. We hypothesize that two key events shaped the early history of the Beijing genotype: (1) its Upper Palaeolithic origin in the Homo sapiens sapiens K-M9 cluster in Central Asia, and (2) primary Neolithic dispersal of the secondary Beijing NTF::IS6110 lineage by Proto-Sino-Tibetan farmers within east Asia (human O-M214/M122 haplogroup). The independent introductions of the Beijing strains from east Asia to northern Eurasia and South Africa were likely historically recent, whereas their differential dissemination within these areas has been influenced by demographic and climatic factors.     

Hypersensitivity to Forcipomyia taiwana (biting midge): clinical analysis and identification of major For t 1, For t 2 and For t 3 allergens

BACKGROUND: Forcipomyia taiwana is a tiny, blood-sucking midge that cause intense pruritis and swelling in sensitive individuals. It is distributed island-wide in rural Taiwan and Southern China. Objective: This study aimed to study the allergic immune responses and identify F. taiwana allergens. METHODS: Crude whole body F. taiwana extracts were prepared with phosphate-buffered saline. The specific IgE antibody was determined by enzyme-linked immunoassay and immunoblotting. Protein was analyzed by electrospray ionization tandem mass spectrometry. RESULTS: Among the 372 subjects that were exposed to F. taiwana bites, 179 (48%) reported an immediate skin reaction with/without delay reaction and 41(11.1%) reported a solely delay reaction. The skin of 21 subjects was tested with F. taiwana extract. Of these 21 subjects, 12 (57.1%) produced immediate skin reactions and contained high levels of specific IgE antibody against F. taiwana. Immunoblotting revealed that 11 allergenic components are able to bind specific IgE. Allergens of 22, 24, 35, 36, and 64 kDa bound 50, 50, 75, 66.7, and 75% of IgE-containing sera tested, respectively. Tryptic fragments of the 24, 35, 36, and 64 kDa allergens were analyzed by ESI-MS/MS. Selected tryptic peptides of 24, 35, and 36, and 64 kDa allergens exhibited significant sequence identity with triosephosphate isomerase of Anopheles merus,Tenebrio molitor,Ochlerotatus togoi, and Chrysops vittatus, fructose 1,6-bisphosphate aldolase of Antheraea yamamai and Homalodisca coagulata, and a slow muscle myosin S1 heavy chain of Homarusamericanus and a protein with unknown function from A. gambiae, respectively. The 35 and 36 kDa proteins may represent different isoforms of the fructose 1,6-bisphosphate aldolase. CONCLUSION: We conclude that immediate reaction to F. taiwana bites is IgE mediated and the 24 (For t 1), 35 (For t 2), and 64 kDa (For t 3) proteins are candidates for major F. taiwana allergens. Further studies are needed to confirm these allergens.     

Substrates for coincidence detection and calcium signaling for induction of synaptic potentiation in the neonatal visual cortex

Regulation of the efficacy of synaptic transmission by activity-dependent processes has been implicated in learning and memory as well as in developmental processes. We previously described transient potentiation of excitatory synapses onto layer 2/3 pyramidal neurons in the visual cortex that is induced by coincident presynaptic stimulation and postsynaptic depolarization. In the adult visual cortex, activation of N-methyl-d-aspartate (NMDA) glutamate receptors is necessary to induce this plasticity. These receptors act as coincidence detectors, sensing presynaptic glutamate release and postsynaptic depolarization, and cause an influx of Ca(2+) that is necessary for the potentiation. In the neurons of the neonatal visual cortex, on the other hand, coincident presynaptic stimulation and postsynaptic depolarization induce stable long-term potentiation (LTP). In addition, reduced but significant LTP can be induced in many neurons in the presence of the NMDA receptor (NMDAR) antagonist, 2-amino-5-phosphonovaleric acid despite the Ca(2+) requirement. Therefore there must be an alternative postsynaptic Ca(2+) source and coincidence detection mechanism linked to the LTP induction mechanism in the neonatal cortex operating in addition to NMDARs. In this study, we find that in layer 2/3 pyramidal neurons, release of Ca(2+) from inositol trisphosphate (InsP(3)) receptor-mediated intracellular stores and influx through voltage-gated Ca(2+) channels (VGCCs) provide alternative postsynaptic Ca(2+) sources. We hypothesize that InsP(3)Rs are coincidence detectors, sensing presynaptic glutamate release through linkage with group I metabotropic glutamate receptors (mGluRs), and depolarization, through VGCCs. We also find that the downstream protein kinases, PKA and PKC, have a role in potentiation in layer 2/3 pyramidal neurons of the neonatal visual cortex.     

Improved PCR sensitivity for direct genotyping of Chlamydia trachomatis serovars by using a nested PCR.

Successful amplification of omp1 DNA by PCR is crucial in the genotyping of Chlamydia trachomatis when directly performed with clinical samples (J. Lan, J. M. M. Walboomers, R. Roosendaal, G. J. van Doornum, D. M. McLaren, C. J. L. M. Meijer, and A. J. C. van den Brule, J. Clin. Microbiol. 31:1060-1065, 1993). Several primers flanking the four variable domains of the omp1 gene were selected and tested for sensitivity in several nested PCRs with serial dilutions of serovar G. The optimal sensitivity obtained was 0.1 to 0.01 inclusion-forming units, similar to that obtained in the C. trachomatis plasmid PCR. With this approach, any C. trachomatis PCR-positive sample can be typed.     

黑尾蜡嘴雀原纹状体发声控制核团的传入性纤维联系

将HRP微电泳人黑尾蜡嘴雀(Eophona mihratoria)端脑原纹状体粗核内,观察其传入性纤维联系.在下列部位发现有密集的逆行标记细胞:(1):原纹状体带核;(2)上纹状体腹侧尾核;(3)新纹状体前部大细胞核外侧部;(4)脑桥蓝斑.以上标记细胞均出现在同侧.结果表明,原纹状体粗核接受原纹状体带核、上纹状体腹侧尾核、新纹状体前部大细胞核外侧部及脑桥蓝斑的传入性投射.     

Atrial sympathetic and parasympathetic nerve sprouting and hyperinnervation induced by subthreshold electrical stimulation of the left stellate ganglion in normal dogs

BACKGROUND: Subthreshold electrical stimulation of the left stellate ganglion (LSG) can induce nerve sprouting and sympathetic hyperinnervation in canine ventricles. It is unclear whether a similar neural plasticity involving both sympathetic and parasympathetic innervation also exists in the atria. METHODS AND RESULTS: We applied subthreshold electrical stimulation at 20 Hz (0.45 ms pulse width) or 5 Hz (1.9 ms pulse width) to the LSG in 6 normal mongrel dogs. After 41+/-9 days, the hearts were harvested and the right and left atrium stained for synaptophysin (SYN), growth-associated protein 43 (GAP43), sympathetic nerve markers tyrosine hydroxylase (TH), and parasympathetic marker choline acetyltransferase (ChAT). Tissues from 6 additional healthy dogs were used as controls. The hearts from dogs with LSG electrical stimulation had a higher density of nerve structures immunopositive to the SYN, GAP43, TH, and ChAT (P<.01) in both right and left atria. Nerve density was equal in right and left atria. There were more TH-positive nerve structures than ChAT-positive nerve structures (P<.01) for both right and left atria. No atrial arrhythmia was observed at the second surgery. CONCLUSIONS: Continuous subthreshold electrical stimulation to the LSG induces both sympathetic and parasympathetic hyperinnervation in both right and left atria in normal dogs.     

Personality of seasonal affective disorder analyzed by Tri-dimensional Personality Questionnaire

BACKGROUND: Although there have been numerous reports in personality of mood disorders, there have been few reports in regard with personality of winter seasonal affective disorder (SAD). Furthermore, no reports have been published concerning summer SAD personality characteristics. Thus, this study was conducted to assess the personality of winter and summer SAD using Tri-dimensional Personality Questionnaire (TPQ) that have been used in a variety of mental disorders. METHODS: A total of 6135 Japanese were evaluated with TPQ, the Seasonal Pattern Assessment Questionnaire (SPAQ) and the Self-rating Depression Scale (SDS). Winter, summer and non-SAD groups were classified by SPAQ. We compared the difference of personality trait among these three groups in consideration of gender, age and SDS score influence. RESULTS: Winter SAD demonstrated higher "Novelty Seeking" and "Harm Avoidance"; summer SAD showed higher "Harm Avoidance" than the non-SAD group. "Harm Avoidance" in both SAD groups was re-analyzed using SDS score as a covariate, and "Novelty Seeking" in winter SAD using age as a covariate. As a result, the significance of high "Novelty Seeking" and high "Harm Avoidance" in winter SAD was excluded. However, "Harm Avoidance" remained the significant difference between summer and non-SAD. LIMITATION: SAD was diagnosed only by SPAQ and not by interview. The state-dependency of "Harm Avoidance" was not confirmed in identical patients over lapse of time. CONCLUSION: Patients with winter SAD have high "Harm Avoidance" dependent on the depressive state that is in accordance with non-seasonal depression. Patients with summer SAD have high "Harm Avoidance" possibly independent from the depressive state.