Volume 117  ·   Issue 11  ·  2004

Background Although there are several drugs and drug combinations for the treatment of Pneumocystis carinii (P. carinii) pneumonia, all drugs have the toxicity as well as low efficacy. Iron chelators have been proposed as a source of new drugs for combating these infections. We hypothesized that iron chelators would suppress the growth of P. carinii by deprivation of the nutritional iron re quired for growth. In this study, a short-term axenic culture system of P. carinii was established. Daphnetin (7,8-dihydroxycoumarin), a known iron chelator, was demonstrated to exhibit in vitro activity against P. carinii in this system. Methods P. carinii organisms were obtained from the lungs of immunosuppressed rats. The culture system consisted of Iscove Dulbecco Eagle' s Minimum Essential Medium (IMDM), supplemented with S-adenosyl-L-methionine, N-acetylglucosamine, putreseine, L-cysteine, L-glutamine, 2-mercaptoethanol, and fetal bovine serum, and was maintained at 37 ℃, in 5 % CO2, 95 %O2, at the optimal pH of 8. 0. The culture system was used to assess the effect of daphnetin on the proliferation of P. carinii organisms. The ultrastructures of the treated organisms were observed by transmission electron microscopy. Results The number of cysts and trophozoites increased 8- to 9-fold and 11- to 12-fold, respectively, after 10 days of culture. Daphnetin was found to suppress the growth of P. carinii in a dose-dependent manner at concentrations between 1 μmol/L and 20 μmol/L. The inhibitory activity was suppressed by the chelation of daphnetin with ferrous sulfate in a 2 1: molar ratio, but it was not suppressed by mixing the culture medium with magnesium sulfate. Reduction of P. carinii numbers after treatment with daphnetin correlated with morphological changes in the organisms, as determined by transmission electron microscopy. Conclusions Daphnetin can suppress the growth of P. carinii in vitro. The efficacy of daphnetin in suppressing the the growth of P. carinii in vitro is related to its ability to chelate iron. Chin Med J 2004; 117(11): 1704-1708

Background A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj). Methods A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients. Results Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1. 07 X106 individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti- Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The re suits of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60 %and 37 %in acute and chronic patients, respectively, with a specificity of 90 %. When the two specific scFvs were combined, their sensitivity was found to be 75 %and 57 %in acute and chronic patients, respectively, with a specificity of 85 %. Conclusions The re sults indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine de sign. Chin Med J 2004; 117 (11): 1697-1703

Background Successful periodontal regeneration depends on the migration, proliferation and differentiation of periodontal ligament cells in periodontal defects. The total protein content and the ultra structure demonstrate the metabolizability and activity of periodontal ligament cells. This study was conducted to observe the effects of Shuanghuangbu, a mixture of medicinal herbs, on the total protein content and the ultra structure of human periodontal ligament cells. Methods Periodontal ligament cells were grown to confluence and then cultured in Dulbecco's modified eagle medium (DMEM) supplemented with Shuanghuangbu over the concentration range of 0 to 1000 μg/ml. The total protein content in cultured cells was determined by using Coommasie brilliant blue technique. Periodontal ligament cells were incubated in 0 and 100 μg/ml Shuanghuangbu decoction for 5 days, then observed through transmission electron microscope. Results The total protein content of human periodontal ligament cells increased in each experiment group added 10 - 1000 μg/ ml Shuanghuangbu respectively, and the effect of 100 μg/ ml was excellent. Under the transmission electron microscope, there were more rough endoplasmic reticulums and mitochodrias in the experiment group than those in the control group. Conclusion Shuanghuangbu stimulates the protein synthesis of human periodontal ligament cells and improves human periodontal ligament cells' metabolizability and activity. Chin Med J 2004; 117(11): 1693-1696

Background Notch activation leads to transcriptional suppression of lineage-specific genes, inhibiting differentiation in response to inductive signals. The Notch signal system contains three parts: Notch molecules, Notch ligands and effectors. Delta4 is a newly-discovered Notch ligand which has received the attention of few detailed studies. This study sought to explore the biological function of Delta4 and observe its effects on 32D cell differentiation. Methods DeIta4-expressing vector pTracer. CMV. Delta4. FLAG was constructed using molecular biological techniques. CHO cells stably transfected with pTracer. CMV. Delta4. FLAG were confirmed to have a Delta4 protein band via Western blotting. High-expression Delta4-CHO clones were selected for the following functional studies. Notchl-CHO and Notch2-CHO were used as host cells. After transiently transfecting with transition protein 1 (TP1), Delta4 activity was compared in both cell lines by means of luciferase analysis. CHO cells were incubated with Notchl-32D cells that had been transfected with Notchl and were observed for granulocyte colony-stimulating factor (GCSF)-induced differentiation. Jagged2-CHO and Delta4-CHO cells transfected with the Notch ligands Jagged2 and Delta4, respectively, were incubated with Noteh1-32D cells to observed inhibition of Notch on GCSF-induced differentiation. Results The vector pTracer. CMV. Delta4. FLAG was constructed successfully. CHO cells were stably transfected with the vector pTracer. CMV. Delta4. FLAG. Two CHO cell lines expressing Delta4 at high levels were selected for use in the study. Delta4 was found to induce signal activity via both Notchl and Notch2 and the induction of signaling activity was stronger in Notch2 cells than in Notchl cells. Compared with other Notch ligands, Delta4 was slightly weaker than Jagged2, but stronger than Delta1 and Jagged 1 in terms of Notchl ligands. In terms of Notch2, Delta4 had a strong signaling activity, but was weaker than Deltal, Jaggedl, and Jagged2. Jagged2 could inhibit Notchl-32D cell differentiation induced by GCSF, but Delta4 could not. Conclusions Delta4 induces both Notchl and Notch2 activity and is a ligand for both of them. The effect of Delta4 is stronger on Notch2 than that on Notchl. Jagged2 can inhibit Notchl-32D cell differentiation induced by GCSF, but Delta4 cannot. Chin Med J 2004; 117(11): 1687-1692

Background Intractable epilepsy may be due to multidrug resistance induced by conventional antiepileptic drugs. The phenomenon is sometimes associated with an overexpression of multidrug resistance gene 1 (MDR1). The purpose of this study was to determine if the overexpression of MDR1 could be induced in astrocytes from rat brains in vitro using antiepileptic drugs. Methods Astrocyte cell cultures from postnatal Wistar rats (within 24 hours of birth) were established. Different concentrations of the antiepileptic drugs phenytoin, phenobarbital, carbamazepine, and valproic acid were added to the cultures for 10, 20, or 30 days. The expression of P-glycoprotein (Pgp), the protein product of MDR1, was investigated with immunocytochemistry. Results Less than 5 % of normal, untreated astrocytes had detectable Pgp staining at any time point. Phenytoin, phenobarbital, carbamazepine, and valproic acid induced the overexpression of Pgp in astrocytes in a dose- and time-dependent manner. Significantly higher levels of Pgp staining were detected at therapeutic concentrations of certain antiepileptic drugs (20 μg/ ml phenobarbital, 40 μg/ ml phenobarbital, and 20 μg/ ml phenytoin) on day 30. Upregulation of Pgp was detected when using higher concentrations of phenytoin, phenobarbital, and valproic acid on day 20 and when using higher concentrations of any of the four antiepileptic drugs on day 30. Conclusions Treatment with antiepileptic drugs may contribute to the overexpression in astrocytes of MDR1 and its protein product, Pgp. The mechanism leading to MDR must be considered in patients undergoing long-term treatment with antiepileptic drugs. Chin Med J 2004; 117 (11): 1682-1686

Background Previous studies have indicated that thrombin (TM) may play a major role in brain edema after intracerebral hemorrhages (ICHs) . However, the mechanism of TM-induced brain edema is poorly understood. In this study, we explored the effect of TM on the permeability of the blood brain barrier(BBB) and investigated its possible mechanism, aiming at providing a potential target for brain edema therapy after ICHs. Methods TM or TM + cathepsin G (CATG) was stereotaxically injected into the right caudate nucleus of Sprague-Dawley rats in vivo. BBB permeability was measured by Evans-Blue extravasation. Brain water content was determined by the dry-wet weight method. Brain microvascular endothelial cells were then cultured in vitro. After TM or TM + CATG was added to the endothelial cell medium, changes in the morphology of cells were dynamically observed by phase-contrast light microscopy, and the expression of matrix metalloproteinase-2 (MMP-2) protein was measured by immunohistochemical method. Results BBB permeability increased at 6 hours after a TM injection into the ipsilateral caudate nucleus(P<0.05), peaked between 24 hours (P<0.01) and 48 hours (P <0. 05) after the injection, and then declined. Brain water content changed in parallel with the changes in BBB permeability. However, at all time points, BBB permeability and brain water content after a TM+CATG injection were not significantly different from the respective parameters in the control group (P> 0. 05) . TM induced endothelial cell contraction in vitro in a time-dependent manner and enhanced the expression of MMP-2 protein. After incubation with TM + CATG, cell morphology and MMP-2 expression did not change significantly as compared to the control group (P>0.05). Conclusions Increased BBB permeability may be one of the mechanisms behind TM-induced cerebral edema. TM induces endothelial cell contraction and promotes MMP-2 expression by activating protease activated receptor-1 (PAR-1), possibly leading to the opening of the BBB. Chin Med J 2004; 117 (11): 1677-1681

Background Previous studies have suggested that interrupted clearance of nuclear DNA-protein complexes after cell death might initiate and propagate systemic lupus erythematosus (SLE). Deoxyribonuclease I (DNase I) may be responsible for the removal of DNAfrom nuclear antigens at sites of high cell turnover, thus preventing the onset of SLE. The purpose of this study was to genotype the single nucleotide polymorphisms (SNPs) of DNase 1 and characterize its gene expression and alternatively spliced transcripts in Chinese patients with SLE in order to understand the pathogenic role of DNase 1 in human SLE. Methods Four SNPs located at the 3' end of the DNase 1 gene, as listed on the SNP website, were selected for analysis. Those SNPs with relatively high heterozygosity were chosen for genotyping in 312 Chine se SLE families using the Taqman minor groove binder (MGB) allelic discrimination method. Haplotypes were constructed and linkage disequilibrium tests were performed using GeneHunter. DNase 1 mRNA expression was detected using real-time polymerase chain reaction (PCR), and alternatively spliced transcripts were isolated using capillary electrophoresis. Any effects the specific SNP haplotypes had on DNase 1 gene expression and the alternatively spliced transcripts were also assessed. Results rs179982 and rs1053874 had high heterozygosity, about 0. 5 in this Chine se cohort, while rsl059857 was also found to be heterozygous. Analysis of the haplotype combining rsl79982-rsl030874 (C-G) and rsl79982-rsl030874-rsl059857 (C- G-G) revealed a skewed transmission in favor of affected offspring. DNase 1 gene expression was higher in SLEpatients thanin normal controls (P<0. 001), but this was not related to disease activity or SNP haplotype. Capillary electrophoresis revealed that the pattern of alternatively spliced transcripts inpatients differed from that of normal controls. Furthermore, different SNP haplotype combinations generated different transcript patterns in SLEpatients. Conclusions The SNP haplotypes are in linkage disequilibrium in Chine se SLE patients and may induce the disease through a modification of DNase 1 mRNA splicing rather than at the level of mRNA expression. There is a relatively unique transcript band in SLE patients independent of special haplotype, which suggests that other unknown factors might be involved in adjusting gene expression. Chin Med J 2004; 117 (11): 1670-1676

Background Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs. Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation. SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immuno histochemical staining. Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc. Conclusion The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation. Chin Med J 2004; 117 (11): 1665-1669

Background A number of reports based on both animal experiments and clinical investigations have pointed out that total parenteral nutrition (TPN) suppresses the function of the exocrine pancreas. Even though pancreatic hypotrophy and dysfunction resulting from TPN may be explained by several mechanisms, the clinically most important cause is that nutrients in circulation affect pancreatic secretion. The effect of nutrients on the exocrine pancreas is still controversial. The aim of the present study was, therefore, to clarify the irfluence of intravenous amino acids and typertonic glucose in TPN solution on the exocrine pancreas. Methods Three mixed TPN solutions, consisting of 30 %or 50 % glucose or of 14 %amino acids, were employed. Twenty-four male Sprague-Dawley rats were randomly divided into four groups, six rats in each group, including a control group and one group receiving each of the three TPN solutions. All animals were killed after 10 days of TPN. Body weight, pancreatic content, and enzyme levels in the pancreas were measured. Results Compared with the control group, pancreatic wet weight was lower in all TPN groups. Glucose significantly decreased the content and concentration of pancreatic protein, but amino acids did not alter the concentration of protein. The level of amylase was lower in all parenterally fed groups, with a greater decrease in the groups treated with amino acids and 30 %glucose than with 50 %glucose. Trypsin levels in all group s receiving TPN were markedly higher than in the control group. Conclusion TPN re suits in atrophy of the pancreas, but trypsin levels increase with TPN treatment. Gluco se elevates the amylase level in the pancreas, while amino acids suppress pancreatic amylase. Amino acids used as a source of protein maintain normal pancreatic protein levels. Chin Med J 2004; 117 (11): 1659-1664

Background Isolated dextrocardia is a rare phenomenon and usually associated with multiple cardiac anomalies. This study was to evaluate the accuracy of diagnosis of isolated dextrocardia by using angiocardiography and to compare it with the re sults of surgery. Methods The clinical data of 27 cases of congenital isolated dextrocardia were collected to understand the diagnostic approaches to the major cardiac anomalies. All cases underwent angiocardiography followed by palliative or curative surgery. The diagnosis was compared by angiocardiography relying on segmental analysis with the pathological features observed in surgery. Results The results of angiocardiography of 22 patients were the same as the pathological features observed during surgery, including one case with congenital left ventricular diverticulum was inadvertently omitted in angiocardiograhy. There were significantly dissimilar diagnoses between angiocardiograhy and post-operation in 5 patients, including anatomical corrected transposition of great arteries misinterpreted as corrected transposition of the great arteries in 1, complete transposition of great arteries misinterpreted as corrected transposition of the great arteries in 1, single ventricle misinterpreted as double-outlet right ventricle in 1, and anatomical double-outlet left ventricle misinterpreted as corrected transposition of the great arteries in 2. Msdiagnostic rate of angio c ardio grahy was almost 20 %. Conclusions Angiocardiography is of great significance in the diagnosis and classification of isolated dextrocardia. However, because of the intricacy of cardiac anomalies of isolated dextrocardia, atrial angiography and double oblique projection are needed to improve the accuracy of diagnosis to support surgical treatments. Chin Med J 2004; 117 (11): 1655-1658