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In this paper, the controllable band width, resolution and sample loading were investigated by the injection system of free-flow electrophoresis (FFE) with gratis gravity. Two general injection methods were described herein. The first method was the one in which sample injection fluxes were variable with constant background flux, while the second was the one in which the background fluxes were flexible with stable sample flux. With methyl green and crystal violet as two viewable model compounds, a series of experiments were performed, and the experimental results revealed that (1) the sample band width could be under desiring control through the regulation of ratios between sample and background fluxes, (2) the separative resolution could be also adjusted elaborately via the regulation of flux ratios during the separation of methyl green and crystal violet with only one charge disparity, and (3) the sample loading could be conveniently controlled via the flux ratios and an approximate maximum sample loading could be selected under the condition of just completed separation of two adjoining solutes. In addition, it was observed that the flux ratio had soft influence on the separative resolution of two solutes. These results were of significance to the designs on band width, resolution and sample loading in the newly developed FFE device with gratis gravity., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

A novel formulation process via co-spray drying ibuprofen (IBU) with mesoporous SBA-15 submicron particles exhibited excellence in production of stable amorphous IBU with significantly enhanced dissolution rate. With drug loading of IBU/SBA-15 ratio being 50:50 (w/w) or below, most drug molecules were entrapped inside the straight mesoporous channels via the co-spray drying and the morphology of SBA-15 submicron particles remained unchanged. IBU confined inside the mesoporous structure was in the amorphous state shown by PXRD and DSC measurements. The amorphous state of IBU in the solid dispersion showed remarkable stability when subject to stress test condition of 40 degrees C/75% RH in open pans for 12 months. The uniform pore walls were believed to prevent the re-crystallization of the homogeneously dispersed drug molecules inside the mesoporous channels with confined nanospace. The dissolution rate of IBU from the co-spray-dried solid dispersion was significantly enhanced to achieve a rapid release. Even after the accelerated stability test, the rapid drug release property was well preserved., (2009 Wiley-Liss, Inc. and the American Pharmacists Association)

Tracheal glomus tumor is an extremely rare neoplasm resected mostly by open surgery or through rigid bronchoscopy. We report two cases presenting with polypoid masses arising from the tracheal membrane in the posterior wall of the lower tracheal segment. The tumor was removed by high-frequency electrocautery and flexible bronchoscopic argon-plasma coagulation, and follow-up bronchoscopy and chest CT did not reveal tumor recurrence 12 months after the operation. In patients with tracheal glomus tumor who have poor surgical tolerance or are not willing to receive an open surgery, flexible bronchoscopic tumor removal can be a good alternative to relieve the airway obstruction symptoms.

Objective: To study the expression and clinical significance of kidney injury molecule-1 (KIM-1) in primary and metastatic renal epithelial neoplasms., Methods: A total of 136 cases of kidney neoplasms were retrospectively reviewed including 63 primary clear cell renal cell carcinomas (RCCs), 22 papillary RCCs, 13 chromophobe RCCs, 7 oncocytomas, 7 RCCs associated with Xp11.2 translocation/TFE3 gene fusions and 24 metastatic clear cell RCCs. Immunostaining for KIM-1 and kidney-specific-protein (Ksp)-cadherin were performed and the relationship to tumor stage and grade in clear cell RCCs was investigated., Results: Expression of KIM-1 was detected in 77.8% (49/63) of clear cell RCCs, 90.9% (20/22) of papillary RCCs, 1/13 of chromophobe RCCs, 7/7 of RCCs associated with Xp11.2 translocation/TFE3 gene fusions and 87.5%(21/24) of the metastatic RCCs, but not detected in 7 cases of oncocytomas. A diffuse expression of KIM-1 was more frequently observed in Furhman nuclear grade III/IV clear cell RCCs (P = 0.010). Ksp-cadherin expression was mainly observed in chromophobe RCCs and oncocytomas., Conclusions: KIM-1 is a specific biomarker for injuried kidney proximal tubules and the corresponding neoplasms, and has a high specificity and sensitivity for primary or metastatic clear cell RCCs, papillary RCCs and RCCs associated with Xp11.2 translocation/TFE3 gene fusions. Combination of KIM-1 and Ksp-cadherin immunostaining can lead to a more precise histological classification of primary kidney epithelial neoplasms and improve the diagnostic accuracy of metastatic RCCs.

Alveolar soft part sarcoma (ASPS) is a rare malignant soft tissue tumor, which rarely occurs in bone. We present a case of ASPS in a 23-year-old man with a 2-month history of back pain. Computed tomography scanning and magnetic resonance images demonstrated a destructive process in the 12th thoracic vertebra associated with a unilateral soft tissue mass. The tumor showed evidence of hypervascularity on MRI; it obviously was enhanced on T1-weighted images after injection of Gd-GDPA, and signal voids were shown on all pulse sequences which may help to differentiate ASPS from other tumors of the vertebra. We believe that this is the first case of ASPS arising in a vertebra.

Objective: To study the prognostic significance of grading system for stromal invasion in pathologic tumor stage T1 (pT1) adenocarcinoma of lung., Methods: Eighty-five cases of surgically resected pT1 lung adenocarcinoma with clinicopathologic and follow-up data were retrospectively reviewed. The degree of invasive growth was classified into three grades according to its location in the tumor. The clinicopathologic characteristics and prognostic significance were analyzed., Results: Amongst the 85 cases studied, 17 cases (20%) were in grade 1, 12 (14%) in grade 2 and 56 (66%) in grade 3. The tumor size was smaller and lymphovascular permeation was less frequently encountered in cases with grade 1 stromal invasion than in those with grade 3 (P=0.005 for tumor size and P=0.018 for occurrence of lymphovascular permeation). The rate of lymph node metastasis and pathologic staging in cases with grade 1 and grade 2 were similar and were significantly lower than those with grade 3 (P=0.007 for rate of lymph node metastasis in grade 1 versus grade 3 tumors, P=0.002 for pathologic stage in grade 1 versus grade 3 tumors, P=0.027 for rate of lymph node metastasis in grade 2 versus grade 3 tumors and P=0.021 for pathologic stage in grade 2 versus grade 3 tumors). There was no statistically significant difference with respect to age, gender and smoking history of the patients, amongst cases in different grades. The overall five-year survival rate was 63%. The five-year survival rates for cases with grade 1, grade 2 and grade 3 were 100%, 83.3% and 46.6%, respectively. The difference between cases with grade 2 and grade 3 was statistically significant (P=0.027). The death rate during follow-up for cases with grade 1, grade 2 and grade 3 were 0, 16.7% and 42.9%, respectively. The difference between cases with grade 1 and grade 3 was statistically significant (P=0.001). Univariate analysis showed that grade of stromal invasion (P=0.001), pathologic stage (P<0.001), presence of lymphovascular permeation (P<0.001) and lymph node involvement (P<0.001) represented important prognostic factors. Multivariate analysis also showed that pathologic stage (P<0.001) was an independent prognostic factor., Conclusions: The grading system of stromal invasion in pulmonary adenocarcinoma correlates with tumor prognosis and other prognostic factors. It represents a useful criterion in prognostic categorization of pT1 adenocarcinoma of lung.

This paper describes a novel free-flow electrophoresis (FFE), which is joined with gratis gravity, gas cushion injector (GCI) and self-balance collector instead of multiple channel pump, for the purpose of preparative purification. The FFE was evaluated by systemic experiments. The results manifest that (i) even though one-channel peristaltic pump is used for the driving of background buffer, there is still stable flow in the FFE chamber; (ii) the stable flow is induced by the gravity-induced pressure due to the difference of buffer surfaces in the GCI and self-balance collector; (iii) the pulse flow of background buffer induced by the peristaltic pump is greatly reduced by the GCI with good compressibility of included air; (iv) the FFE can be well used for zone electrophoretic separation of amino acids; (v) up to 20 inlets simultaneous sample injection and up to five to tenfold condensation of amino acid can be achieved by combining the FFE device with the method of moving reaction boundary. To the best of authors' knowledge, FFE has not been used for such separation and condensation of amino acids. The relevant results achieved in the paper have evident significance for the development of preparative FFE.

Objective: To carry out a genetic detection and analysis of Von Hippel-Lindau (VHL) gene in Chinese patients with sporadic pheochromocytoma., Methods: DNA samples were extracted from peripheral blood cells and fresh pheochromocytoma specimens from 41 patients with sporadic pheochromocytoma were assayed by polymerase chain reaction and direct sequencing. The DNA samples of 50 healthy volunteers were extracted from peripheral blood as a control. The PCR products of exon 1, exon 2 and exon 3 were used for molecular analysis of the VHL gene. The genetic detection of family members of VHL gene mutations was also performed., Results: One of mutations was located at nucleotide 572 (G-->C) in exon 2, presenting a codon 120 from arginine (R) to threonine (T). Tow small insertions were locatated at nucleotide 623T (TTTGTtG) in exon 2, leading to a frameshift mutation. There were also three carriers of G572C and three carriers of 623T (TTTGTtG) in family members of the three cases., Conclusion: There are some Chinese patients with sporadic pheochromocytoma with tumorigenic VHL gene mutations. It is recommended to use the genetic detection and analysis of VHL gene as a routine examination for patients with sporadic pheoehromoeytoma under the age of 50 years with questionable family history. The genetic detection and analysis of VHL gene may be useful as a marker for the diagnosis of hereditary pheochromocytoma.

Objective: The epidemic data of Wenchuan area earthquake trauma of ear, nose and throat were investigated., Methods: Affected families and sufferers of part of disaster area in Sichuan province in 2008 were investigated with emphasis on severity, cause and treatment of otorhinolaryngologic trauma., Results: Two hundred and twenty-seven cases of otorhinolaryngologic trauma in survival crowd 3 days after earthquake were included in this study. Most of them were minor trauma or complex lesion. There were 185 cases of soft tissue wound in head and face, 13 cases of fracture of the nose, 18 cases of hemorrhage of the nose, 7 cases of fracture of the skull base, 4 cases of lacerated wound of the auricle. Diagnosis and management were carried out by medical aid post and field ambulance on different condition. The methods of treatment included debridement and suture of soft tissue wound and positioning of fracture. If there was serious injury and accompanied complex lesion, the patients should be sent to hospital after emergent treatment. Forty-six cases were treated in medical aid post, except 3 cases of complex lesion by transportation, 43 cases recovered in 5 to 10 days after treatment. One hundred and eighty-one cases were treated in field ambulance, except 3 cases with fracture of skull base by transportation. Among 31 hospitalized patients, 26 recovered and were discharged before 26th of may, 5 were still in ward because of complex lesion. Among 147 cases treated out of wards with soft tissue wound, 146 cases recovered and bone fracture in rehabilitation. 1 case of lacerated wound of auricle was infected with delayed healing. There were no complication in all cases., Conclusions: For earthquake trauma of the ear, nose and throat, emergent management are debridement, suture and positioning of fracture. For seriously injured patient, transportation to hospital in time can decrease complications and death rate.

Objective: To analyze the causes of benign central airway stenoses and to evaluate the efficacy of interventional treatments through flexible bronchoscopy., Methods: Three hundred and eighty-six outpatients and inpatients with benign central airway stenoses in our hospital from January 1999 to December 2006 were retrospectively analyzed. Interventional treatments through flexible bronchoscopy were used to treat the benign central airway stenoses. The endoscopic interventional treatments included laser, electrocautery, argon plasma coagulation, cryotherapy, balloon dilation and stent insertion. Airway diameters, FEV1 and dyspnea index of patients were evaluated before and immediately after the last treatment procedure., Results: The main causes of benign central airway stenoses were as follows: tuberculosis in 64.25% (248/386), secondary to prolonged orotracheal intubation or tracheotomy in 15.03% (58/386), injury in 3.63% (14/ 386) and inhalation burns in 3.11% (12/386), others were 54 cases. All the 386 patients received endoscopic interventional treatments. 89.89% (347/386) of the patients experienced improvement in dyspnea and cough. The average airway diameter increased from (2.49 +/- 1.57) mm to (6.41 +/- 1.70) mm (t = 47.427, P < 0.01). Dyspnea index decreased from 2.40 +/- 0.79 to 0.64 +/- 0.50 (t = 44.226, P < 0.01). The average value of FEV1 evaluated in 115 inpatients increased from (2.11 +/- 0.60) L to (3.46 +/- 0.75) L (t = 20.128, P < 0.01). Most patients needed multiple interventional treatments except 26 patients who received a single endoscopic treatment. Stable control of the diseases was achieved in 65.54% (253/ 386) patients 3 months after the last operation., Conclusion: Tuberculosis is the most common cause of benign central airway stenoses in this series. Utilization of interventional methods through flexible bronchoscopy is effective in treating benign central airway stenoses.

Objective: To report the diagnostic experience for broncholithiasis, and to evaluate the value of flexible bronchoscopic management for the disease., Methods: The clinical data, radiological features, bronchoscopy findings, the effect and complications of bronchoscopic management in 31 patients with broncholithiasis admitted to changhai hospital of second military medical university and zhejiang Taizhou Hospital between 2000 and 2007 were retrospectively reviewed., Results: There were 19 men and 12 women, the mean age was 58.6 +/- 12.6. There were 26 cases missed diagnosis, and 19 cases were misdiagnosis. The diagnosis was confirmed by chest CT combined with bronchoscopy in all the patients. Thirty-six broncholiths were revealed, of which 26 were intraluminal, 9 eroding in the bronchial wall, and 1 peribronchial. In 26 patients, 31 broncholiths were managed by flexible bronchoscope, and 25 broncholiths were successfully removed. The success rate for intraluminal free broncholiths was higher (23/25). One patient experienced hemoptysis of 300 ml after therapy, but no significant complications occurred in other patients., Conclusions: The diagnosis of broncholithiasis can be easily missed or misdiagnosed. Chest CT combined with bronchoscopy were useful methods for the diagnosis of the disease. Extraction of broncholiths with flexible bronchoscope is a safe and effective management, especially for intraluminal broncholiths.

Objective: To detect the VHL gene mutations in a Chinese family with nonsyndromic pheochromocytoma., Methods: Mutations of VHL gene were detected in a Chinese family with nonsyndromic pheochromocytoma. Five patients and fifteen relatives were involved in this study. Peripheral blood was collected and total genomic DNA was prepared for polymerase chain reaction (PCR). PCR products of all the three exons of VHL gene were purified and a direct gene sequence analysis was performed., Results: All the five patients presented a codon 125 from Histidine (H) to Proline (P) change at nucleotide 587 (A --> C) in exon 2. Seven members of fifteen relatives were carriers with the same VHL gene mutation. Two carriers were detected with bilateral adrenal tumors and right renal cyst respectively by ultrasonic inspection., Conclusion: The novel VHL gene mutation detected in this kindred may be the causative gene. Genetic test can detect the carriers in an early period. It is recommended as a routine method of genetic test in nonsyndromic pheochromocytoma patients.

Objective: To describe the clinical features and treatment of relapsing polychondritis with involvement of the respiratory tract., Method: Thirteen cases (admitted from Aug 2000 to Oct 2006) of relapsing polychondritis with involvement of the respiratory tract treated in our hospital were retrospectively analyzed., Results: There were 9 males and 4 females, with ages ranging from 30 to 61 years (mean 50 years). At early stage of the disease, clinical manifestations included cough, throat pain and hoarseness. Patients in later stage usually complained of chest distress, shortness of breath and dyspnea. Severe complications were repeated lower respiratory tract infections and/or respiratory failure. Bronchoscopic examination revealed an edematous larynx, narrowing of the glottis, tracheobronchial edema, turgescence of bronchial cartilage rings and airway stenosis at early stage. At later stage of the disease, malacia of trachea and bronchi due to disappearance of bronchial cartilage rings, and partly obliteration of the both trachea and main bronchus on expiration were demonstrated. Thoracic CT scan, with three-dimensional reconstruction of the airways, demonstrated a diffusely thickened tracheobronchial wall with tracheobronchial stenosis in earlier period of the disease and showed severe narrowing of both trachea and main bronchi in later period. Lung function measurements showed a moderate obstructive ventilatory disorder in 5 patients. Medical treatment with corticosteroids and immunosuppressive drugs was given in 12 patients. Symptoms were improved in 6 patients in earlier period of the disease, however, 6 patients in later period were not relieved. Twelve self-expanding metallic stents were placed in the airways (trachea and/or main bronchi), and obstruction of the respiratory tract was relieved in 5 patients, but there was no improvement in 1 patient who later was treated with positive airway pressure support. The 13 patients were followed for 1 to 48 months, and 12 patients survived, but one patient died 2 years after diagnosis., Conclusions: Corticosteroid therapy is effective in improving the symptoms and delaying the progression of relapsing polychondritis with involvement of the respiratory tract at early stage. At later stage of the disease, airway interventional therapy, such as metallic stent placement, tracheostomy or positive airway pressure support, can be used to treat airway obstruction and to improve the survival.

Objective: EphB1 is a member of the Eph family of receptor tyrosine kinases that is involved in embryonic nervous and vascular system development. Over- or underexpression of certain Eph receptors has been found in some cancer samples compared to normal tissue. Expression of Eph receptors is related to malignant transformation, metastasis, differentiation, and prognosis of cancers. Recently, the EphB subfamily has been shown to be involved in the tumorigenesis of colorectal cancer. In the present study, expression of the EphB1 transcript and protein in gastric carcinoma samples was determined to investigate the roles of EphB1 in development, progress and prognosis of gastric carcinoma., Methods: Quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemical staining were used., Results: The EphB1 transcript was overexpressed in 68.9% (42/61) and underexpressed in 14.8% (9/61) of cases. However, the expression of protein was greatly different from the transcript expression, with overexpression and underexpression being 17.2% (10/58) and 44.8% (26/58), respectively. In addition, we showed that underexpression of EphB1 protein is significantly associated with invasion, stage and metastasis in gastric carcinomas., Conclusion: EphB1 may have a tumor-suppressive role in gastric cancer., ((c) 2008 S. Karger AG, Basel)

Objective: To observe the occurrence of restenosis after metallic stents implantation in benign tracheal and main bronchial stenosis, and to evaluate the therapeutic effects of balloon dilatation, cryotherapy and electrocautery., Methods: Thirty patients with benign tracheal stenosis and 35 patients with main bronchial stenosis caused by tuberculosis were treated with metallic stents implantation and a follow-up was carried out to observe the occurrence of restenosis. Combined balloon dilatation, cryotherapy and electrocautery were used to manage the restenosis. The lumen of stenotic segment, dyspnea index and pulmonary function were measured before management and after the patient's condition became stable., Results: Restenosis occurred in 6 of the 30 patients receiving tracheal stenting and in 8 of the 35 patients receiving bronchial stenting, the restenosis rates were 20% and 22.86%, respectively. In total, 30 Chinese-made stents and 36 Ultraflex stents were implanted, and the restenosis rate was 20% and 22.2%, respectively (P > 0.05). The restenosis rate was 4/9 in the upper segmental tracheal stents, and was 9.09% in the middle-lower segmental stents (chi(2) = 5.114, P < 0.05, but chi(c)(2) = 3.100, P > 0.05). The restenosis rate was 16.67% in the fibrotic stage of endobronchial tuberculosis (EBTB), and was 60% in the inflammatory reaction stage of EBTB (chi(2) = 4.564, P < 0.05, but chi(c)(2) = 2.437, P > 0.05). The effective rate was 4/6 in the tracheal stenting restenosis patients, and was 2/4 and 2/2 in the upper and middle-lower segment groups, respectively. The effective rate was 7/8 in the bronchial stenting restenosis patients. After management, the lumen diameter of stenotic segment in the tracheal stenting restenosis patients increased from (3.33 +/- 1.63) mm to (9.33 +/- 3.98) mm (P < 0.02), the dyspnea index decreased from (3.67 +/- 0.52) to (1.50 +/- 1.64) (P < 0.02), the vital capacity (VC) increased from (1.39 +/- 0.17) L to (2.43 +/- 0.70) L (P < 0.01), the forced expiratory volume in one second (FEV(1)) increased from (1.02 +/- 0.15) L to (2.00 +/- 0.72) L (P < 0.02). After management, the lumen diameter of stenosis segment in the bronchial stenting restenosis patients increased from (3.00 +/- 0.76) mm to (7.38 +/- 2.00) mm (P < 0.001), the dyspnea index decreased from (1.63 +/- 0.52) to (0.50 +/- 0.76) (P < 0.005), VC increased from (1.74 +/- 0.16) L to (2.74 +/- 0.41) L (P < 0.001), FEV(1) increased from (1.41 +/- 0.19) L to (2.36 +/- 0.37) L (P < 0.001)., Conclusion: Restenosis occurred in some patients with benign tracheal and tuberculous bronchial stenosis after metallic stent implantation. The restenosis rate was higher in the upper tracheal stenosis than that in the middle-lower tracheal stenosis. Caution should be taken when metallic stents are placed in this part of the trachea. The restenosis rate was higher in the inflammatory reaction stage of EBTB than in the fibrotic stage. Effort should be made to avoid placing metallic stents at this stage of the disease. Balloon dilatation, cryotherapy and eletrocautery are effective methods in managing restenosis after stent implantation.

The crystal structure of beta-luffin at 2.0 A resolution was solved by the molecular-replacement method using polyalanyl trichosanthin as the search model. The structure was refined with CNS1.1, giving R(work) = 0.162 and R(free) = 0.204. The r.m.s.d.s of the bond lengths and bond angles are 0.008 A and 1.3 degrees, respectively. The overall structure is similar to those of other type I RIPs. Three N-acetylglucosamine (Nag) molecules are linked to residues Asn2, Asn78 and Asn85 of the protein.

Three mutant crystals of neo-trichosanthin (n-TCS), R163K, R163H and R163Q, were obtained by the hanging drop vapor diffusion method. Structure determination indicated that there are no significant differences between the mutants and n-TCS except in the active pocket. All of them were also soaked in sodium citrate buffer (pH 4. 5) containing 20% KCl and 10 mg/ml AMP. Structure determination suggests that in the active pocket of the crystals of R163K and R163H, parallel to the aromatic ring of Tyr70, each mutant possesses an adenine. The relationship between structure and function is discussed. Biochemical analysis reveals that the mutants R163K and R163H have N-glycosidase activity, while R163Q does not. This suggests that R163 is a crucial residue for the enzyme activity of n-TCS, and its role is providing proton.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. TCS has long been used as an abortifacient in China. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have attracted more and more attention. An isoform of trichosanthin, neo-trichosanthin (n-TCS), has been cloned and expressed as recombinant protein. The biochemical studies revealed that n-TCS has virtually the same rRNA N-glycosidase activity as TCS. The crystal structure of n-TCS is similar to TCS. The crystal of Y70A n-TCS, the mutant of recombinant n-TCS, was soaked in sodium citrate buffer (pH 5.5) containing 25% KCl and AMP (10 mg/ml) prior to data collection. After structure determination and refinement, no electron density corresponding to adenine can be detected around the active pocket. Furthermore, the reaction products of Y70A n-TCS and AMP incubated at various reaction times were analyzed using HPLC. No adenine can be detected. These results suggest that Tyr70 is crucial to n-TCS for its substrate recognition, binding and perhaps N-glycosidase activity.

Amino acids Tyr14 and Arg22 in trichosanthin are residues on helix A1 close to the active-site cleft. They are invariant in various type-I and type-II ribosome-inactivating proteins. In this study, Tyr14 was changed to Phe and Arg22 to Lys and Leu. Modified proteins were purified, and activities compared by assaying their median inhibitory concentration (ID50) on a rabbit-reticulocyte-lysate protein-synthesis system. While the ID50 of wild-type trichosanthin was 0.02 nM, those for [Phe14], [Lys22], [Leu22] and [Phe14, Leu22]trichosanthin were 0.10, 0.03, 0.25 and 0.15 nM, respectively. Therefore, compared with Tyr14, Arg22 appears to play a more important role in trichosanthin. Structural studies on [Leu22]trichosanthin showed that two water molecules occupy the space left by the side chain of Arg22, and hydrogen bonds exist between these water molecules and nearby residues to retain the conformation. The use of intermolecular rather than intramolecular hydrogen bonds may have an adverse effect on stability or folding of the protein and results in a mild decrease in activity.

A fundamental problem in biochemistry and molecular biology is understanding the spatial structure of macromolecules and then analyzing their functions. In this study, the three-dimensional structure of a ribosome-inactivating protein luffin-alpha was predicted using a neural network method and molecular dynamics simulation. A feedforward neural network with the backpropagation learning algorithm were trained on model class of homologous proteins including trichosanthin and alpha-momorcharin. The distance constraints for the C alpha atoms in the protein backbone were utilized to generate a folded crude conformation of luffin-alpha by model building and the steepest descent minimization approach. The crude conformation was refined by molecular dynamics techniques and a simulated annealing procedure. The interaction between luffin-alpha and its analogous substrate GAGA was also simulated to understand its action mechanism.

Trichosanthin is a protein used medicinally in China for abortifacient purposes. It is also an RNA N-glycosidase which inactivates eukaryotic ribosomes by removing adenine4324 from 28S rRNA. Site-directed mutagenesis was performed to probe the role of Gln156, Glu160 and Glu189 in the active site of trichosanthin. The purified altered proteins were assayed for their potency in inhibiting in vitro protein synthesis. The data indicate Glu160 is involved in the catalytic reaction. Kinetics studies suggest the carboxylate group of Glu160 serves to stabilize the transition-state complex. Similar to ricin A, the variant [E160A]trichosanthin is more potent than [E160D]trichosanthin. This is because Glu189 serves as a back-up of the carboxylate group in case Glu160 is mutated to alanine. However, removal of Glu189 in the presence of Glu160 does not affect the ID50 value drastically. An activity of 1800-fold less than that of the wild-type protein was found when both Glu160 and Glu189 were changed to alanine, indicating that some other residues in the active site are also taken part in the lowering of energy barrier for the catalytic reaction. Although Gln156 is highly conserved in related proteins, its mutation to alanine only slightly decreases the activity, showing that this residue does not participate directly in catalysis.

An X-ray reflection data set of the orthorhombic crystal of Trichosanthin (TCS) at 1.73 A resolution had been collected using the area detector. We determined TCS crystal structure by the molecular replacement method using the data of the known alpha-momocharin model and TCS intensities, and then TCS structure refinement at 1.73 A resolution was performed with the restrained least-squares refinement. A final R-factor of 0.186 was obtained with a model obeying standard geometry within 0.013 A in bond lengths and 2.48 degrees in bond angles. The final model contains 133 solvent molecules in the asymmetric unit. This paper gives a detailed description of TCS molecule structure, temperature factors, hydrogen bonds, bound water, the distribution of conserved residues and the interactions between the TCS molecules. The hydrogen bond between the hydroxyl group of conserved residue 14Tyr and O157 plays an important role in maintaining the active site conformation. Conserved residue 160Glu and its conformation at the active site play a key role in the catalytic activity.

Crystals of alpha-momorcharin were obtained by vapour phase diffusion. The crystal belongs to the space group R3, with unit cell constants a = b = 131.3 A, c = 39.5 A. There is one alpha-momorcharin molecule in the asymmetric unit; a data set at 3 A has been collected.

A series of novel organocyclophosphorus compounds including twenty-three 2,3-disubstituted-2,3-dihydro-4H-1,3,2-benzoxazaphosphorin-4-one-2 -sulfide (compounds I) and three 2,3-disubstituted-2,3-dihydro-4H-1,3,2-benzoxazaphosphorin-4-th ion e-2-oxide (compounds II) have been synthesized. Their structures were determined by IR, UV, 1HNMR, X-ray diffraction and elemental analysis. Each of compounds I showed a strong absorption between 1290-1319 cm-1 in the IR spectrum. Comprehensive studies of the spectral data and experimental results indicated that this strong characteristic absorption is different from that of the P = O linkage and thus could be considered as a result of the stretching vibration of (unsaturated C)-N single bond. In the course of synthesis of the compounds, an abnormal product (Formula: see text) is frequently formed as an isomer of the expected product I (Formula: see text). However, by suitable choice of condensation conditions, either of the isomers could be made predominantly in yield and be readily isolated from the other by recrystallizing for several times from appropriate solvent. Data for the melting point, IR, UV and 1HNMR spectra of three pairs of the isomers are given, and primary analyses of the spectra are made as well.

A model completed recently of two trichosanthin molecules in an asymmetric unit belonging to the monoclinic system is reported. It can be seen that the molecular structure consists of two domains, one large and one small, and that there are some features in the secondary structure. The dissimilarity between two molecules as well as their interactions in an independent unit correlated in a non-symmetric way has been described, moreover, the binding sites of the heavyatom position in the derivatives are also discussed.

Comparison of the crystal structure of a complex of the phage 434 Cro protein and a synthetic DNA operator with the complex of the same operator and the 434 repressor DNA-binding domain shows different DNA conformations in the two structures. Binding of the protein determines the precise conformation of the DNA in each case.