BACKGROUNDSARS-CoV is the causative agent of severe acute respiratory syndrome (SARS) which has been associated with outbreaks of SARS in Guangdong, Hong Kong and Beijing of China, and other regions worldwide. SARS-CoV from human has shown some variations but its origin is still unknown. The genotyping and phylogeny of SARS-CoV were analyzed and reported in this paper.

METHODSFull or partial genomes of 44 SARS-CoV strains were collected from GenBank. The genotype, single nucleotide polymorphism and phylogeny of these SARS-CoV strains were analyzed by molecular biological, bioinformatic and epidemiological methods.

RESULTSThere were 188 point mutations in the 33 virus full genomes with the counts of mutation mounting to 297. Further analysis was carried out among 36 of 188 loci with more than two times of mutation. All the 36 mutation loci occurred in coding sequences and 22 loci were non-synonymous. The gene mutation rates of replicase 1AB, S2 domain of spike glycoprotein and nucleocapsid protein were lower (0.079% - 0.103%). There were 4 mutation loci in S1 domain of spike glycoprotein. The gene mutation rate of ORF10 was the highest (3.333%) with 4 mutation loci in this small domain (120 bp) and 3 of 4 loci related to deletion mutation. By bioinformatics processing and analysis, the nucleotides at 7 loci of genome (T:T:A:G:T:C:T/C:G:G:A:C:T:C) can classify SARS-CoV into two types. Therefore a novel definition is put forward that according to these 7 loci of mutation, 40 strains of SARS-CoV in GenBank can be grouped into two genotypes, T:T:A:G:T:C:T and C:G:G:A:C:T:C, and named as SARS-CoV Yexin genotype and Xiaohong genotype. The two genotypes can be further divided into some sub-genotypes. These genotypes can also be approved by phylogenetic tree of three levels of 44 loci of mutation, spike glycoprotein gene and complete genome sequence. Compared to various strains among SARS-CoV Yexin genotype and Xiaohong genotype, GD01 strain of Yexin genotype is more closely related to SARS-CoV like-virus from animals.

CONCLUSIONThe results mentioned above suggest that SARS-CoV is responding to host immunological pressures and experiencing variation which provide clues, information and evidence of molecular biology for the clinical pathology, vaccine developing and epidemic investigation.

Evolution, Molecular ; Genome, Viral ; Genotype ; Phylogeny ; Point Mutation ; SARS Virus ; genetics

BACKGROUNDHuman urotensin II (UII) is the most potent mammalian vasoconstrictor identified so far. Our previous study showed that UII is a potent mitogen of airway smooth muscle cells (ASMC) inducing ASMC proliferation in a dose-dependent manner. The signal transduction pathway of UII mitogenic effect remains to be clarified. This study was conducted to investigate the signal transduction pathway in the proliferation of ASMC induced by UII.

METHODSIn primary cultures of rat ASMCs, activities of protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and calcineurin (CaN) induced by UII were measured. The effect of CaN on PKC and MAPK was studied by adding cyclosporin A (CsA), a specific inhibitor of CaN. Using H7 and PD98059, inhibitors of PKC and MAPK, respectively, to study the effect of PKC and MAPK on CaN. The cytosolic free calcium concentration induced by UII was measured using Fura-2/AM.

RESULTSUII 10(-7) mol/L stimulated ASMC PKC and MAPK activities by 44% and 24% (P < 0.01), respectively, after incubating for 20 minutes. It increased CaN activity in a time-dependent manner, being 1.68 times as that of control for 24 hours (P < 0.01). It promoted the cytosolic free calcium concentration increase of 18% (P < 0.01). CsA 10(-6) mol/L and H7 50 micromol/L inhibited UII-stimulated CaN activity by 45% (P < 0.01) and 21% (P < 0.05), respectively, while PD98059 50 micromol/L had no effect on CaN activity (P > 0.05). CsA 10(-6) mol/L inhibited UII-stimulated PKC activity by 14% (P < 0.05), while having no effect on MAPK activity (P > 0.05).

CONCLUSIONSUII increases cytosolic free calcium concentration and activates PKC, MAPK and CaN. The signal transduction pathway between PKC and CaN has cross-talk.

Animals ; Calcineurin ; metabolism ; Cells, Cultured ; Enzyme Activation ; Mitogen-Activated Protein Kinases ; metabolism ; Mitogens ; pharmacology ; Myocytes, Smooth Muscle ; cytology ; Protein Kinase C ; metabolism ; Rats ; Signal Transduction ; physiology ; Trachea ; cytology ; Urotensins ; pharmacology

BACKGROUNDAirway smooth muscle proliferation plays an important role in airway remodeling in asthma. But little is known about the intracellular signal pathway in the airway smooth muscle cell proliferation in asthma. The objective of this paper is to investigate the contribution of protein kinase C (PKC) and its alpha isoform to passively sensitized human airway smooth muscle cells (HASMCs) proliferation.

METHODSHASMCs in culture were passively sensitized with 10% serum from asthmatic patients, with non-asthmatic human serum treated HASMCs used as the control. The proliferation of HASMCs was examined by cell cycle analysis, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. The effect of PKC agonist phorbol 12-myristate 13-acetate (PMA) and PKC inhibitor Ro-31-8220 on the proliferation of HASMCs exposed to human asthmatic serum and non-asthmatic control serum was also examined by the same methods. The protein and mRNA expression of PKC-alpha in passively sensitized HASMCs were detected by immunofluorescence staining and reverse transcription-polymerase chain reaction.

RESULTSThe percentage of S phase, absorbance (value A) and the positive percentage of PCNA protein expression in HASMCs passively sensitized with asthmatic serum were (16.30 +/- 2.68)%, 0.430 +/- 0.060 and (63.4 +/- 7.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [(10.01 +/- 1.38)%, 0.328 +/- 0.034 and (37.2 +/- 4.8)%, respectively] (P < 0.05). After HASMCs were passively sensitized with asthmatic serum, they were treated with PMA, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (20.33 +/- 3.39)%, 0.542 +/- 0.065 and (76.0 +/- 8.7)% respectively, which were significantly increased compared with asthmatic serum sensitized HASMCs without PMA(P < 0.05). After HASMCs passively sensitized with asthmatic serum were treated with Ro-31-8220, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (11.21 +/- 1.56)%, 0.331 +/- 0.047 and (38.8 +/- 6.0)% respectively, which were significantly decreased compared with asthmatic serum sensitized HASMCs without Ro-31-8220 (P < 0.05). The relative ratio of value A of PKC-alpha mRNA and the positive percentage of PKC-alpha protein expression in passively sensitized HASMCs were 1.23 +/- 0.10 and (61.1 +/- 9.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [1.05 +/- 0.09 and (34.9 +/- 6.7)%, respectively] (P < 0.05).

CONCLUSIONSThe proliferation of HASMCs passively sensitized with human asthmatic serum is increased. PKC and its alpha isoform may contribute to this proliferation.

Asthma ; immunology ; pathology ; Cell Division ; physiology ; Cells, Cultured ; Humans ; Immunization, Passive ; Myocytes, Smooth Muscle ; pathology ; physiology ; Protein Kinase C ; physiology ; Protein Kinase C-alpha ; Signal Transduction ; physiology

BACKGROUNDAsthma is clinically related with the degree of eosinophilic inflammation. How asthmatic airway inflammation is affected is still poorly understood. So the effects of bone marrow-derived hematopoietic cells expressing CD(34) (CD(34)(+)) and interleukin-5 (IL-5) receptor messenger RNA (IL-5R mRNA+) on asthmatic airway inflammation were investigated.

METHODSBalb/c mice were sensitized and challenged by ovalbumin (OVA) to establish an asthmatic model while control mice were sensitized and exposed to sterile saline. The mice were killed at different time points after being challenged by OVA and sterile saline. Then, bronchoalveolar lavage fluid (BALF), peripheral blood (PB) and bone marrow (BM) were prepared. Eosinophils in PB (PBEOS) and BALF (BALFEOS), nuclear cells in BALF, PB and BM were counted. By flow cytometry, the percentage of CD(34)(+) cells to nucleated cells in PB, BM and the relative number of CD(34)(+) cells in PB (PBCD(34)(+)) and BM (BMCD(34)(+)) were calculated. Immunocytochemistry and in situ hybridization were used to investigate the hematopoietic cells with co-localized expression of CD(34) and IL-5R mRNA in BM (BMCD34+IL-5R mRNA+). The percentage of BMCD34+IL-5R mRNA+ to BMCD(34)(+) was calculated.

RESULTSTwelve hours after challenge by OVA, BALFEOS and PBEOS in the experimental group were significantly higher than those in the control group (P < 0.01). Twenty-four hours after OVA challenge, BALFEOS, PBEOS and BMCD34+IL-5R mRNA+ were elevated maximally, significantly different from those in the control group (P < 0.01). Forty-eight hours after OVA challenge, BALFEOS and BMCD34+IL-5R mRNA+ were still significantly higher than those of the controls (P < 0.01). The other markers reverted to normal. In 60 mice, BMCD34+IL-5R mRNA+ was closely correlated with the BALEOS, PBEOS, BMCD(34)(+) and BMCD(34)(+) (%) (P < 0.05).

CONCLUSIONSThe amount of CD(34)(+) cells expressing IL-5R mRNA increased in the BM of asthmatic model mice, which favors eosinophilopoiesis and eosinophilic airway inflammation. A signal pathway exists between the lungs and the bone marrow, which is involved in the initiation and maintenance of asthmatic airway inflammation.

Animals ; Antigens, CD34 ; analysis ; Asthma ; immunology ; Bone Marrow Cells ; cytology ; Bronchoalveolar Lavage Fluid ; cytology ; Inflammation ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; analysis ; Receptors, Interleukin ; genetics ; Receptors, Interleukin-5

BACKGROUNDChronic hypoxia can cause pulmonary hypertension and pulmonary heart disease with high mortality. The signal transduction pathway of protein kinase C (PKC) plays an important role in chronic pulmonary hypertension. So it is necessary to investigate the effect of PKC on voltage-gated potassium (K+) channels in pulmonary artery smooth muscle cells of rats exposed to chronic hypoxia.

METHODSMale Wistar rats were randomly divided into a control group (group A) and a chronic hypoxia group (group B). Group B received hypoxia [oxygen concentration (10 +/- 1)%] eight hours per day for four consecutive weeks. Single pulmonary artery smooth muscle cells were obtained using an acute enzyme separation method. Conventional whole cell patch clamp technique was used to record resting membrane potential, membrane capacitance and voltage-gated K+ currents. The changes in voltage-gated K+ currents before and after applying paramethoxyamphetamine (PMA) (500 nmol/L), an agonist of PKC, and PMA plus carbohydrate mixture of glucose, fructose and xylitol (GFX) (30 nmol/L), an inhibitor of PKC, were compared between the two groups.

RESULTSThe resting membrane potential in group B was significantly lower than that of group A: -(29.0 +/- 4.8) mV (n = 18) vs -(42.5 +/- 4.6) mV (n = 35) (P < 0.01). But there was no change in membrane capacitance between the two groups: (17.9 +/- 4.6) pF (n = 40) vs (19.7 +/- 5.8) pF (n = 31) (P > 0.05). The voltage-gated K+ currents were significantly inhibited by PMA in group A, and this effect was reversed by GFX. However, the voltage-gated K+ currents in group B were not affected by PMA.

CONCLUSIONSThe resting membrane potential and voltage-gated K+ currents in pulmonary artery smooth muscle cells from rats exposed to chronic hypoxia decreased significantly. It seems that PKC has different effects on the voltage-gated K+ currents of pulmonary artery smooth muscle cells under different conditions.

Animals ; Chronic Disease ; Hypoxia ; physiopathology ; Male ; Myocytes, Smooth Muscle ; physiology ; Potassium Channels, Voltage-Gated ; physiology ; Protein Kinase C ; physiology ; Pulmonary Artery ; physiopathology ; Random Allocation ; Rats ; Rats, Wistar

BACKGROUNDSevere acute respiratory syndrome (SARS) is characterized by both an atypical pneumonia and efficient nosocomial transmission. However, it remains unknown whether the infectivity and the virulence of the pathogen will change throughout the successive transmission. This study was conducted to compare the clinical features and management regimens of patients with SARS among the multiple generations from nosocomial transmission initiated by a super-spreader.

METHODSThe clinical data of 84 epidemiologically-linked SARS patients from a hospital outbreak were retrospectively studied. All patients, in whom a clear-cut transmission generation could be noted, had a direct or indirect exposure to the index patient and the epidemic successively propagated through the multiple generations of cases within a short period of time.

RESULTSThere were 66 women and 18 men with mean age of (29.2 +/- 10.3) years in this cluster; and 96.4% of whom were health care workers. Detailed contact tracing identified 35 (41.7%) first-generation cases, 34 (40.5%) second-generation cases, and 15 (17.8%) third-generation cases. No statistical differences among the multiple generations of transmission were found in terms of age, gender, incubation period and length of hospital stay. With the advanced transmission generations, the initial temperature lowered, the number of cases with dry cough decreased. There were no statistical differences in the peak temperature and duration of fever, other accompanying symptoms, leucopenia; however, the time from initial pulmonary infiltrates to radiographic recovery shortened (P < 0.05). No differences were found in maximum number of lung fields involved, duration from the onset of fever to the occurrence of pulmonary infiltrates and time from the initial pulmonary infiltrate to its peak among the multiple transmission generations (P > 0.05). No statistical differences were found in modes of oxygen therapy and sorts of antibiotics prescribed among the various transmission generations (P > 0.05); however, as with the advanced transmission generations, the number of cases prescribed with methylprednisolone, human gamma-globulin, interferon-alpha, antiviral drugs (oral ribavirin or oseltamivir) increased (P < 0.05) and time from admission to starting these medication shortened (P < 0.05).

CONCLUSIONSThere is no evidence that SARS infection will evolve or transmit within a fashion that permits it to become less powerful throughout the successive transmission within a short time.

Adult ; Contact Tracing ; Cross Infection ; physiopathology ; Female ; Humans ; Male ; Personnel, Hospital ; Retrospective Studies ; Severe Acute Respiratory Syndrome ; physiopathology ; transmission

BACKGROUNDMedically unexplained dyspnea occurs commonly in medical settings and remains poorly understood. This study was conducted to investigate the psychophysiological characteristics of medically unexplained dyspnea and the efficacy of breathing retraining for these patients.

METHODSA group of patients with medically unexplained dyspnea were compared to patients with a variety of organic lung diseases and healthy subjects. In another group of patients, the influence of breathing therapy on complaints, anxiety, and breath-holding was evaluated for an average of 1.5 years.

RESULTSPatients with medically unexplained dyspnea reported more intense dyspnea than patients with a variety of organic lung diseases. Additionally, they were anxious and presented a broad range of symptoms in daily life and under challenge, for instance voluntary hyperventilation. More than one third of them qualified for panic disorder. They had shorter breath-holding time at rest, less increase in breath-holding time and higher chances of showing a "paradoxical" decrease of breath-holding time after hyperventilation. A combination of PaO2, forced expiratory volume in one second (FEV1), and anxiety measures distinguished them from organic dyspnea. Breathing retraining profoundly improved their symptoms and decreased the level of state and trait anxiety. Moreover, they better tolerated the voluntary hyperventilation and the symptoms induced were also markedly decreased after therapy. Breath-holding time was prolonged and PetCO2 in a representative group of patients increased.

CONCLUSIONSPatients with medically unexplained dyspnea appear to have the feature of a "psychosomatic" patient: an anxious patient with a wide variety of symptoms of different organ systems that do not have an organic basis. They can be distinguished from organic dyspnea using a small set of physiological and psychological measures. Breathing retraining turns out to be an effective therapy for those "difficult to treat patients".

Adult ; Aged ; Anxiety ; complications ; Breathing Exercises ; Dyspnea ; psychology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Panic Disorder ; complications ; Psychophysiologic Disorders

Minimally Invasive Surgical Procedures ; Philosophy, Medical

Anxiety ; Dyspnea ; psychology ; Humans ; Personality

OBJECTIVETo investigate feasible treatment methods for plantar ulcers in leprosy patients according to the agreement between the Ministry of Health (MOH) of China and the Leprosy Mission International (LMI).

METHODSA total of 2599 complicated foot ulcers in 1804 leprosy cases underwent surgic treatment. Plastic fixation and supports were used, dressings were changed regularly, and protective footwear and modified insoles were provided.

RESULTSOf the 2599 foot ulcers 1446 (55.64%) healed. The cure rate of the patients treated in leprosy hospitals was 71.31%, with 219 (15.15%) recurrences of foot ulcers. The recurrence rate of those who lived at home was 18.35%.

CONCLUSIONSComprehensive treatment of foot ulcers has a high cure rate and a low recurrence rate. Reduction of workload, avoidance of long distance walking, intensification of education on foot self-care and provision of financial support are the main measures for preventing a recurrence of foot ulcers.

Foot Ulcer ; therapy ; Humans ; Leprosy ; complications ; Treatment Outcome