1999年 · 第112卷第06期

OBJECTIVE To explore the relationship between apoptosis and spontaneous hypertension, and the effect of angiotensin converting enzyme (ACE) inhibitor on apoptosis. METHODS Male spontaneously hypertensive rats (SHR) and normotensive control rats (WKY) at different ages were used, meanwhile, the treatment of SHR with ramipril, an inhibitor of ACE was administered orally (1 mg.kg-1.d-1) to SHR from 3 to 10 or from 5 to 10 weeks of age. Apoptosis in cardiomyocytes of SHR was quantified by a maximal labeling (Lmax) method and the characteristic features of apoptosis were identified by electron microscopy (EM), in situ labeling of DNA strand breaks with terminal deoxynucleotidyl transferase mediated dUTP end labeling (TUNEL) and autoradiographic analysis of DNA fragments. RESULTS The RESULTS of a quantitative method showed an age-dependent increase in apoptosis in the cardiac tissues of SHR. A significant increase in DNA breaks occurred as early as 4 weeks and continued to increase up to a plateau at 16 weeks in the cardiac tissue of SHR whereas there was no significant change in apoptosis in WKY up to 64 weeks. Moreover, after the treatment of SHR with ramipril, an inhibitor of angiotensin converting enzyme (ACE), from 3 to 10 or from 5 to 10 weeks of age, the DNA fragmentation as well as blood pressure (BP) was reduced significantly compared with that of untreated SHR (P<0.01), and similar to that of the control WKY. CONCLUSIONS There is a significant increase in the apoptosis of SHR cardiac tissues with increasing age, and ramipril can significantly prevent the increase of apoptosis and in blood pressure, which demonstrates that apoptosis may be involved in the pathogenesis of genetic hypertension. The inhibition of apoptosis as well as hypertension by ACE inhibitors may open a new avenue for developing therapeutic approach for hypertension.

OBJECTIVE To investigate the effect of lithium on the growth of bone marrow stromal cells. METHODS We compared the different effects of lithium with the concentration of 0.1-12 mmol/L on the growth of murine fibroblast colony forming unit (mCFU-F), human fibroblast colony forming unit (hCFU-F) and their intercolonial cells. Pure murine bone marrow stromal cell assay was used to investigate the mechanism. RESULTS Cultures with 1-4 mmol/L lithium resulted in significant increase in the number of hCFU-F, but distinct decrease in the number of mCFU-F. Furthermore, there were much more endothelial cells between mCFU-Fs than those between hCFU-Fs. The direct effect of 1-4 mmol/L lithium on the growth of pure murine fibroblasts and endothelial cells was stimulatory. Serum-free endothelial cell conditioned medium (EC-CM) could inhibit the growth of mCFU-F. CONCLUSIONS 1-4 mmol/L lithium has stimulatory effect on the growth of pure murine fibroblasts and endothelial cells. The inhibitory effect of lithium on mCFU-F formation may result from the effect of increased endothelial cells.

OBJECTIVE To investigate the diagnostic role of antibodies to glutamic acid decarboxylase (GAD65-Ab) in latent autoimmune diabetes of adults (LADA) and the frequency of GAD-Ab in Chinese patients initially diagnosed as non-insulin-dependent diabetes mellitus (NIDDM). METHODS Forty-five control subjects and 195 consecutive inpatients initially classified as NIDDM with ≥ 35 years of age at onset and nonketotic history for >6 months after diagnosis, were recruited. In vitro transcripted and translated recombinant human 35S-GAD65 was used in radioligand assay of GAD-Ab. RESULTS The overall prevalence of GAD65-Ab was 14.8% (29/195) in NIDDM patients and 2.2% (1/45) in control subjects, respectively. Of the 29 GAD65-Ab positive patients, 17 (58.6%) were insulin-deficient while 12 (41.4%) were non-insulin-deficient. The prevalence of GAD65-Ab in NIDDM group with age of<40 years at diabetes onset, ketotic history, body mass index (BMI)<21 kg/m2, were significantly higher than that of corresponding control diabetic subgroups (2.5, 4.1 and 3.2 times, respectively). The sex, duration, symptoms of polyphagia, polydipsia, polyuria and weight loss at onset of the disease were not related to the prevalence of GAD65-Ab positivity. CONCLUSIONS In China, patients initially diagnosed as NIDDM may in many cases suffer from LADA. Testing by GAD65-Ab may be of assistance to identifying LADA at the earliest stage of disease.

OBJECTIVE To investigate the mechanism of cytokine regulation in atopy by analyzing the effects of three major cytokines, IL-4, IL-12 and IFN-gamma on in vitro IgE synthesis of human peripheral blood mononuclear cell (PBMC) from normal individuals and atopic patients. METHODS We investigated 5 normal individuals and 22 atopic patients including 12 allergic asthma, 8 allergic rhinitis and 2 atopic dermatitis. Human PBMCs were separated and incubated in 96-wells culture plates with different cytokines. Cultured for 7 days, the supernatant was collected and the contents of IgE synthesized in vitro were detected. RESULTS There was no IgE synthesis in vitro of PBMC from all the normal individuals and 7 patients, however, other 15 patients' PBMC could produce IgE in vitro. We found that rhIL-4 could induce in vitro IgE synthesis of PBMC from all the normal individuals and 22 patients. rhIL-12 could inhibit IgE synthesis induced by rhIL-4. Further investigation of cytokine effects on IgE synthesis was conducted in 15 patients whose PBMCs did produce IgE in vitro (average level was 714±1105 ng/L). The following effects were observed: (1) 10 μg/L rhIL-4 could augment in vitro IgE synthesis from 714±1105 ng/L to higher level of 1483±1396 ng/L; (2) 20 μg/L rhIL-12 could inhibit in vitro IgE synthesis from 714±1105 ng/L to lower level of 452±969 ng/L; (3) rhIL-12 could block IgE synthesis induced by rhIL-4 to the level of 583±1084 ng/L; (4) 50 μg/L IFN-gamma could inhibit in vitro IgE synthesis from 714±1105 ng/L to the level of 461±1063 ng/L. CONCLUSIONS rhIL-4 could induce in vitro IgE synthesis, rhIFN-gamma could inhibit the in vitro IgE synthesis and rhIL-12 could antagonize rhIL-4 effect on in vitro IgE synthesis of PBMC from atopic patients.

OBJECTIVE To examine whether growth hormone (GH) promotes the phosphorylation of epidermal growth factor (EGF) receptor and to elucidate the mechanisms by which EGF receptors were transactivated by GH stimulation. METHODS Cultured cardiac myocytes were stimulated by GH directly or pretreated with inhibitors before GH stimulation. The phosphorylations of EGF receptor and JAK2 were examined with immunoprecipitation followed by Western blotting using anti-phosphotyrosine antibody (4G10). The activities of extracellular signal-regulated kinases (ERKs) were assayed with the method of MBP-containing gel. RESULTS GH stimulated phosphorylation of EGF receptor in a time-dependent manner. Tyrphostin AG1478, a selective inhibitor of EGF receptor, strongly suppressed GH-induced ERK activation, while tyrphostin AG1295, a selective inhibitor of PDGF receptor, had no effects on the activation of ERKs stimulated by GH in cardiac myocytes. In addition, GH induced tyrosine phosphorylation of JAK2, a cytoplasmic protein tyrosine kinase, in cardiac myocytes. Moreover, tyrphostin B42, an inhibitor of JAK2 suppressed GH-induced phosphorylation of EGF receptor as well as GH-induced activation of ERKs in cardiac myocytes. CONCLUSIONS GH evokes the phosphorylation of EGF receptor in cardiac myocytes through activating JAK2. Phosphorylated EGF receptor plays a critical role in GH signaling pathway leading to ERK activation in cardiac myocytes.

OBJECTIVE To investigate the atherogenic role of very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) fraction isolated from plasma of apolipoprotein E (apoE)-knockout mice. METHODS Containing both VLDL and IDL (apoEko-VLDL/IDL), lipoprotein fraction was isolated from plasma of apoE-knockout mice by ultracentrifugation, and its interaction with J774 macrophages was examined together with its physicochemical properties including lipid content, electrophoresis mobility and apolipoprotein components. RESULTS ApoEko-VLDL/IDL could be actively taken up by J774 macrophages, resulting in marked cholesterol ester accumulation in the cells (55±9 nmol/mg cell protein), which was a 7-fold increase over the corresponding lever induced by low density lipoprotein and significantly higher than non-loaded group (P<0.01). CONCLUSIONS ApoEko-VLDL/IDL induced significant cholesterol ester accumulation through an apoE independent pathway. This pathway may explain, partly the mechanism of cholesterol deposition in arterial wall in apoE-knockout mice.

OBJECTIVE To search differentially expressed sequences correlated with pathogenesis of human nasopharyngeal carcinoma (NPC), including the candidates of tumor suppressor genes. METHODS Representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from normal human primary cultures of nasopharyngeal epithelial cells and cDNA from NPC cell line HNE1. The source of differentially expressed products were proved by Southern blot, Northern blot and in situ hybridization. The fragments were cloned with pGEM-T easy kit and sequenced by the chain termination reaction. RESULTS Four differentially expressed cDNA fragments were isolated in the fourth subtractive hybridization using cDNA from normal human nasopharyngeal epithelial cells as tester amplicon and cDNA from NPC cell line HNE1 as driver amplicon by cDNA RDA. These differential cDNA fragments revealed that they really came from the tester amplicon and were not expressed or down-regulated in the NPC HNE1 cells. Some of the genes were expressed only in human nasopharyngeal epithelial cells but deleted or down-regulated in the biopsies of NPC. Of these obtained clones, some were the sequences of the human known genes including house-keeping genes, the others represented novel gene sequences. CONCLUSIONS The differentially expressed products including the candidates of tumor-suppressor genes may be associated with the initiation of the NPC.

OBJECTIVE To assess the role of human papillomavirus (HPV) in the carcinogenesis of the esophageal carcinomas in a high-incidence geographical area in Northern China. METHODS The presence of HPV types 16 and 18 DNA in the three esophageal carcinoma cell lines was examined by type-specific polymerase chain reaction (PCR). RESULTS No HPV type 16 and 18 DNA was found in any of the three esophageal carcinoma cell lines. CONCLUSIONS The results suggest that the role of HPV in human esophageal carcinogenesis in a high-incidence geographical area remains to be elucidated.

OBJECTIVE To study the genes related to tin miners' lung cancer tissue. METHODS Differential display mRNA. RESULTS Thirty cDNA fragments which differentially expressed in lung cancer tissues and the same patient's normal lung tissues were discovered. Among these, 16 expressed in lung cancer tissues, not in normal lung tissues; fourteen expressed on the contrary. Six cDNA fragment sequence was determined. Five sequences CG2, CG7, CG8, CA5 and CC6 had less than 75% homology with known sequences in GenBank BLAST, so they were believed to be new sequences which we have recorded in Genbank. Only one fragment coded CG3 had homology up to 95% with human ribosome protein L27a gene. CONCLUSIONS mRNA differential display provides a unique and powerful experimental system to study differential gene expression in tin miners' lung cancer tissues and the same patient's normal lung tissues. Using the system, differential expression of 30 cDNA fragments was observed. Six of them may be used to study the molecular mechanism of miners' radon-associated lung cancer.

OBJECTIVE To study the molecular mechanisms of drug resistance in Mycobacterium (M). tuberculosis, to evaluate the value of the beta subunit of RNA polymerase (rpoB), the ribosomal siz protein (rpsL), 16Sr RNA (rrs), catalase-peroxidase gene (katG) genes, and inhA regulatory sequence as genetic markers for rifampin (RFP), streptomycin (SM), isoniazid (INH) resistance, and to develop new methods for detecting the drug resistance. METHODS The rpoB, rpsL, rrs, katG genes, and inhA regulatory sequence in 85 M. tuberculosis isolates were analyzed with polymerase chain reaction (PCR), PCR-single-stranded conformation polymorphism analyses (SSCP), PCR-nucleotide sequence analyses (NS) and PCR-restriction fragment length polymorphism (RFLP). RESULTS The sensitivity of amplifying the drug-resistant genes with PCR was 1-10 pg DNA. Twenty-eight drug-sensitive strains had no alterations in the rpoB, rpsL, rrs, katG genes, and inhA regulatory sequences. 93.3% of 45M. tuberculosis RFP-resistant (RFPr) isolates had rpoB mutations. Codon 531 and 526 of the rpoB are the most common sites of nucleotide substitutions. 72.5% of 40 SM-resistant (SMr) isolates had an identical mutation at codon 43 of the rpsL gene. No isolates had a mutation at codon 88 of the rpsL. Only 7.5% of these SMr isolates had A-to-C transversions at position 513 of the rrs gene. Of 34 INH-resistant (INHr) isolates, 11.8% had complete katG deletions, 55.9% had mutations in the selected region of katG. Only 8.8% had alterations in the inhA regulatory sequences. 60.9% of RFPr, INHr, and SMr isolates had mutations in genetic markers for these drug resistance. CONCLUSIONS Most drug resistance in M. tuberculosis was due to simple mutations occurring in chromosomally encoded genes. Alterations in rpoB, rpsL and katG gene may be the important mechanism of M. tuberculosis resistance to RFP, SM, and INH. PCR, PCR-SSCP, PCR-NS, and PCR-RFLP are going to become the simple, rapid and reliable diagnostic tests for drug resistance in M. tuberculosis.

With the improvement in the living standards of the Chinese people, more attention has been drawn to the importance of dermatology and the need for dermatological service. This fact has resulted in the advancement of dermatology in China in the recent years.