Decrease of galectin-3 in keratinocytes: A potential diagnostic marker and a critical contributor to the pathogenesis of psoriasis.

Psoriasis-specific proteins dysregulated in keratinocytes and involved in the pathophysiological process of psoriasis remains elusive. We report here that epidermal galectin-3 expression is significantly downregulated in lesional skin, but not in non-lesional skin in psoriasis patients, nor in a group of diseases known as psoriasiform dermatitis clinically and histologically similar to psoriasis. The deficiency of epidermal galectin-3 is sufficient to promote development of psoriatic lesions, as evidenced by more severe skin inflammation in galectin-3 knockout (gal3-/-) mice, compared to wild-type mice, after imiquimod treatment, and in skin from gal3-/- mice grafted onto wildtype mice. The development of psoriatic-like lesions is attributable to 1) the spontaneously tuning up of psoriasis signatures in keratinocytes through JNK pathway; and 2) neutrophil accumulation caused by the enhanced leukocyte-recruiting capacity associated with overexpression of S100A7-9 and CXCL-1, 8 in keratinocytes with impaired galectin-3 expression. Psoriasis-like skin inflammation is significantly improved in gal-3-/- mice both by inhibition of neutrophils accumulation with a selective CXCR2 antagonist of SB225002, and by intracutaneous injection of recombinant galectin-3. Overall, these findings offer promising galectin-3-related diagnostic and therapeutic resolutions of psoriasis.

IP3R-Grp75-VDAC1-MCU calcium regulation axis antagonists protect podocytes from apoptosis and decrease proteinuria in an Adriamycin nephropathy rat model.

BACKGROUND: The mechanism of podocyte apoptosis is not fully understood. In addition, the role of the inositol 1,4,5-triphosphate receptor (IP3R)/glucose-regulated protein 75 (Grp75)/voltage-dependent anion channel 1 (VDAC1)/mitochondrial calcium uniporter (MCU) calcium regulation axis, which is located at sites of endoplasmic reticulum (ER) mitochondria coupling, in the mechanism of podocyte apoptosis is unclear. This study aimed to understand the roles of this axis in podocyte apoptosis and explore potential targets for podocyte protection. METHODS: The expression of IP3R, Grp75, VDAC1, and MCU and mitochondrial Ca2+ were analyzed during Adriamycin- or angiotensin II-induced apoptosis in cultured mouse podocytes. The interaction between IP3R, Grp75, and VDAC1 was investigated using co-immunoprecipitation experiments. The effects of IP3R, Grp75, and MCU agonists and antagonists on mitochondrial Ca2+ and apoptosis were investigated in cultured podocytes. The podocyte-protective effects of an MCU inhibitor were further investigated in rats with Adriamycin-induced nephropathy. RESULTS: Increased expression of IP3R, Grp75, VDAC1 and MCU, enhanced interaction among the IP3R-Grp75-VDAC1 complex, mitochondrial Ca2+ overload, and increased active caspase-3 levels were confirmed during Adriamycin- or angiotensin II-induced mouse podocyte apoptosis. Agonists of this axis facilitated mitochondrial Ca2+ overload and podocyte apoptosis, whereas specific antagonists against IP3R, Grp75, or MCU prevented mitochondrial Ca2+ overload and podocyte apoptosis. A specific MCU inhibitor prevented Adriamycin-induced proteinuria and podocyte foot process effacement in rats. CONCLUSIONS: This study identified a novel pathway in which the IP3R-Grp75-VDAC1-MCU calcium regulation axis mediated podocyte apoptosis by facilitating mitochondrial Ca2+ overload. Antagonists that inhibit Ca2+ transfer from ER to mitochondria protected mouse podocytes from apoptosis. An MCU inhibitor protected podocytes and decreased proteinuria in rats with Adriamycin-induced nephropathy. Therefore, antagonists to this pathway have promise as novel podocyte-protective drugs.

[Mutational analysis of the methyl-CpG-binding protein 2 (MECP2) gene in male autism patients].

OBJECTIVE: To investigate mutations in the methyl-CpG-binding protein 2 (MECP2) gene in male autism patients by PCR, denaturing high-performance liquid chromatography (DHPLC) and sequencing to explore the role of mutations in MECP2 in autism patients. METHODS: We recruited DNA samples from 44 male autism patients who matched the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DMS-IV) standards. DHPLC was used to screen the mutations in MECP2 gene, and DNA sequencing was performed for the samples with positive DHPLC results. The family members were further investigated in the patients with missense mutations in MECP2 gene. RESULTS: Four cases were found to have mutations in MECP2 gene, including missense mutations of c.590C>T(T197M)in one case and c.602C>T(A201V)in one case, and synonymous mutations of c.1053C>G in one case and c.897C>T in one case. In addition, we found C>T variation in intron 3 at the +74 bp before exon 4, a SNP (rs2071569) usually detected in Chinese population. In the case with c.602C>T(A201V)mutation, his mother and maternal grandfather had the same mutation. His mother had normal phenotype, but his maternal grandfather had depressive disease. CONCLUSION: Mutations in MECP2 are present in male autism patients with relatively higher prevalence, suggesting that these mutations may play roles in the pathogenesis of autism.

Copy number variation of FCGR3A rather than FCGR3B and FCGR2B is associated with susceptibility to anti-GBM disease.

Anti-glomerular basement membrane antibody disease (anti-GBM disease) is a rare disorder characteristic of universally poor outcome. Fcgamma receptors (FcgammaRs) play important roles in anti-GBM disease based on evidence from animal models. Copy number variation (CNV) influences disease susceptibility. The FcgammaRs genes show CNV, and CNV of the FCGR3B gene is associated with glomerulonephritis in systemic lupus erythematosus and anti-neutrophil cytoplasmic antibody-associated small vasculitis. Here, we investigated CNV of three FCGR genes, including two (FCGR3A and FCGR3B) for activating FcgammaRs and one (FCGR2B) for inhibitory FcgammaR by duplex quantitative real-time PCR. Copy numbers were analyzed by Applied Biosystems CopyCaller Software v1.0. We first demonstrated the distribution of CNV of FCGR3A, FCGR3B and no CNV of FCGR2B in Chinese population (including 47 anti-GBM patients and 146 healthy controls). The frequency of CNV of FCGR3A was observed to be significantly higher than matched healthy controls (27.7 versus 12.3%, P = 0.013, odds ratio 1.21-6.10). Considering previous report about gene knock-out animal models and CNV effect of FCGR3A, we thus propose that CNV in members of FCGR family should have different roles in the pathogenesis of human anti-GBM disease.

[Screening of mitochondrial deoxyribonucleic acid 3271T > C, 8356T > C, 9176T > C/G and 13513G > A mutations in mitochondrial encephalomyopathies].

OBJECTIVE: To investigate the spectrum of mitochondrial DNA (deoxyribonucleic acid) 3271T > C, 8356T > C, 9176T > C/G and 13513G > A mutations in Chinese patients with mitochondrial encephalomyopathies. METHODS: Peripheral blood samples were collected from 500 mitochondrial encephalomyopathies patients clinically diagnosed as mitochondrial encephalomyopathy lactic acidosis & stroke-like episodes (MELAS), myoclonus epilepsy & ragged-red fibers (MERRF) or Leigh's syndrome from October 2005 to October 2009. The methods of PCR- polymerase chain reaction-restriction fragment length polymorphism (RFLP) and PCR-sequencing were performed to identify the mutations. RESULTS: No patients with the 3271T > C, 8356T > C, 9176T > C/G or 13513G > A mutations were identified. CONCLUSION: The mutations of 3271T > C, 8356T > C, 9176T > C/G and 13513G > A are rare causes of mitochondrial encephalomyopathies in Chinese patients.

[Effects of in vivo transfer of human chemokine-like factor 1 gene on cardiac function after acute myocardial infarction in rats].

OBJECTIVE: To study the effects of chemokine-like factor 1(CKLF1)-plasmid transfer on cardiac function in a rat acute myocardial infarction (AMI) model. METHODS: Thirty male SD rats were randomly devided into 3 groups. One hundred micrograms of CKLF1-plasmid, empty plasmid or saline were injected intramuscularly with in vivo electroporation, respectively. Rats were subjected to left coronary artery ligation on the 6th day after gene transfer. Ultrasonic cardiography and hemodynamics were conducted and evaluated on the 22nd day after gene transfer. Then, the animals were sacrificed for determination of percentage of myocardial infarcion. RESULTS: The left ventricular ejection fraction in CKLF1 group (67.02% +/- 12.24%) was significantly higher than that in the saline group (43.64% +/- 7.82%) and empty plasmid group (47.56% +/- 4.10%), P<0.05. Fractional shortening of left ventricle in CKLF1 group (33.83% +/- 10.15%) was higher than that in saline group (18.49% +/- 3.96%) and empty plasmid group (20.85% +/- 2.24%), P<0.05. The maximal velocity of left ventricular pressure ascensus was higher in CKLF1 group [(5 720.01 +/- 826.32) mmHg/s, 1 mmHg=0.133 kPa] than in saline group [(3 955.69 +/- 685.91) mmHg/s] and in empty plasmid group [(4 412.03 +/- 500.74) mmHg/s)], P<0.05. And the maximal velosity of left ventricular pressure descensus was higher in CKLF1 group [(4 636.23 +/- 407.17) mmHg/s] than in saline group [(2 984.82 +/- 615.24) mmHg/s] and in empty plasmid group [(2 963.87 +/- 419.36) mmHg/s], P<0.05. While the percentage of myocardial infarction in CKLF1 group (29.63% +/- 3.93%) was smaller than that in saline group (38.01% +/- 5.48%) and in empty plasmid group (37.50% +/- 6.33%), P<0.05. CONCLUSION: CKLF1 gene transfer can limit the mass of myocardial infarction and improve post-infarction cardiac function.

[Two novel mutations of GJB1 gene associated with typical X-linked Charcot-Marie-Tooth disease].

OBJECTIVE: To analyze the relationship between phenotype and genotype and the role of immune cells in the pathogenesis of X-linked Charcot-Marie-Tooth disease (CMT1X). METHODS: The probands of the two families with X-linked dominant inherited peripheral neuropathy were evaluated clinically, electrophysiologically, pathologically and genetically. The available family members were genetic analyzed and the novel mutations were compared with other known ones. RESULTS: (1) In both families, affected members presented progressive weakness and wasting of distal extremities and it seems that males suffered more severely than affected females with onset in the first decade of their life. Proband of family 1 showed moderately elevated CSF protein and marked increase of IgG-syn in CSF.(2) Nerve conduction velocity (NCV) of the peripheral nerves was intermediately slow in both motor and sensory nerves exhibiting the features of demyelination. Brain-stem auditory evoked potentials (BAEPs) was abnormal in the proband of family 1: delayed I-III interpeak intervals were recorded but with normal III-V interpeak intervals. (3) Sural nerve biopsy in the probands of the two families showed a prominent distinguished loss of myelinated fibers and a few clusters of regenerating axons without conspicuous onion-bulb formations. Thinly myelinated fibers was prominent in family 2 but not in family 1. Immunohistochemical staining showed that there were positive CD68 cells in the endoneurial space and lamellar sheath. (4) By genetic testing, we identified two novel missense mutations of GJB1 gene, which resulted in Ile127Phe amino acid substitution in family 1(located in the intracellular loop of connexin 32) and Asp178Gly amino acid substitution in family 2 (located in the 2(nd) extracellular loop of CX32), respectively. Both mutations were highly conserved in low species and were predicted to be possibly damaging through Polyphen prediction tool. CONCLUSION: The two novel GJB1 gene mutations cause a spectrum of clinical manifestations of CMT1X in both families. However, the mutations site of CX32 alone cannot predict these phenotypic variations in CMT1X fully. The immune system may be involved in the pathogenesis of the disease.

Endogenously generated sulfur dioxide and its vasorelaxant effect in rats.

AIM: The present study was designed to explore the endogenous production and localization of the sulfur dioxide (SO2)/aspartate aminotransferase pathway in vascular tissues of rats and to examine its vasorelaxant effect on isolated aortic rings,as well as the possible mechanisms. METHODS: The content of SO2 in the samples was determined by using high performance liquid chromatography with fluorescence detection. Aspartate aminotransferase activity and its gene expression were measured by an enzymatic method and quantitative RT-PCR, respectively. Aspartate aminotransferase mRNA location in aorta was detected by in situ hybridization. The vasorelaxant effect of SO2 on isolated aortic rings of the rats was investigated in vitro. L-type calcium channel blocker, nicardipine, and L-type calcium channel agonist, Bay K8644, were used to explore the mechanisms by which SO2 relaxed the aortic rings. RESULTS: Aorta had the highest SO2 content among the vascular tissues tested (P<0.01). The aortic aspartate aminotransferase mRNA located in endothelia and vascular smooth muscle cells beneath the endothelial layer.Furthermore, a physiological dose of the SO2 derivatives (Na2SO3/NaHSO3) relaxed isolated artery rings slightly, whereas higher doses (1-12 mmol/L) relaxed rings in a concentration-dependent manner. Pretreatment with nicardipine eliminated the vasorelaxant response of the norepinephrine-contracted rings to SO2 completely. Incubation with nicardipine or SO2 derivatives successfully prevented vasoconstriction induced by Bay K8644. CONCLUSION: Endogenous SO2 and its derivatives have a vasorelaxant function, the mechanisms of which might involve the inhibition of the L-type calcium channel.

[Expression of major histocompatibility complex class II antigens on lungs in rat with bleomycin-induced pulmonary fibrosis].

OBJECTIVE: To explore the expression levels of MHC II molecules and its regulator genes CIITA on bleomycin-induced pulmonary fibrosis in rats, and to investigate the underlying immunologic mechanisms of pulmonary fibrosis. METHODS: The rats were treated with either a single intratracheal bleomycin injection (fibrosis group) or a normal saline injection (control group). The pathologic changes of lung tissues stained with HE and Masson were observed, and the contents of hydroxyproline were detected on the 7th and 28th day respectively after bleomycin administration. The expression of MHC II molecules in the lung tissues was evaluated with immunohistochemistry techniques, and the percentage of MHC II+ cells was measured. The amounts of total CIITA and type I, III and IV CIITA mRNA of lung tissues were measured by real-time PCR using Taqman probe. RESULTS: (1)The percentage of MHC II+ cells in lung tissues increased significantly in fibrosis group compared with that of control group on the 7th day and the 28th day [(0.10 +/-0.03) vs (0.06+/-0.02), P < 0.05; (0.15+/-0.03) vs (0.06+/-0.01),P < 0.01, respectively]; In fibrosis group, the percentage on the 28th day was higher than that on the 7th day [(0.15+/-0.03) vs (0.10+/-0.03), P < 0.05]; (2) Compared with control group on the 7th day, total CIIA mRNA increased 170.4% [(2.89+/-1.07) vs (1.07+/-0.46), P < 0.05], type I CIIA increased 258.8% [(0.77+/-0.38) vs (0.21+/-0.09), P < 0.05], while type IV CIITA decreased 87.2% [(0.39+/-0.15) vs (3.01+/-0.79), P < 0.01]; On the 28th day, total CIITA mRNA increased 98.6% [(4.14+/-1.15) vs (2.08+/-0.76), P < 0.05], type I CIIA increased 137.1% [(0.79+/-0.34) vs (0.33+/-0.23), P < 0.05], type IV CIITA mRNA still decreased, but there was no significant difference [(2.98 +/-0.92) vs (3.95+/-0.93), P > 0.05]; In fibrosis group, type IV CIIA mRNA was 667.3% [(2.98+/-0.92) vs (0.39+/-0.15), P < 0.01] higher on the 28th day than that on the 7th day; Type III CIIA mRNA levels of both groups had no significant difference. CONCLUSION: MHC II/CIITA system of lung tissues was probably involved in the development of rat pulmonary fibrosis.

[Mitochondrial DNA mutation analysis in 97 Chinese patients with mitochondrial cephalomyopathy].

OBJECTIVE: To investigate the characteristics of mitochondrial DNA mutations and genotype-phenotype correlations in mitochondrial encephalomyopathies. METHODS: Biopsy of skeletal muscle and collection of peripheral blood samples were conducted among 97 patients with mitochondrial encephalomyopathies. Southern blotting, PCR-RFLP and direct sequencing of PCR products were performed to search large scale deletions, and common and uncommon pathological point mutations in the muscle and/or blood mtDNA. RESULTS: Seventy-seven patients were identified to be with mitochondrial DNA mutations, including single large deletion (n = 21), multiple large-scale deletions (n = 4), A3243G point mutation (n = 43), A8344G point mutation (n = 6), T8993G mutation (n = 1), T8993C mutation (n = 1), and T3271C mutation (n = 1). Total mtDNA sequencing revealed 4 different novel point mutations in four unrelated patients with isolated mitochondrial myopathy. CONCLUSION: The type and frequency of mtDNA mutations in this series of Chinese mitochondrial encephalopathies are consistent with those reported abroad, Although there is some association between the genotype and phenotype, heterogeneity in phenotype and genotype is also a prominent feature seen in this series of patients, especially those with A3243G mutation.

[Identification of an ideal noninvasive method to detect A3243G gene mutation in MELAS syndrome].

OBJECTIVE: To identify a better non-invasive method to detect the carrier of mitochondrial A3243G mutation, a cause of mitochondrial encephalopathy-lactic acidosis-stroke like episode (MELAS) syndrome. METHODS: DNA was extracted from the peripheral blood, urine, hair follicle, and saliva of 25 MELAS syndrome patients carrying A3243G mutation and their mothers and other maternal relatives, 33 persons in number, and the muscle tissues from 5 patients obtained by biopsy. A3243G mutation was detected by PCR-RFLP method, and the A3243G mutation ratio was identified by measuring the density of each band and calculation with the software AlphaEase 5.0. RESULTS: A3243G mutations were detected in all tissues of the 25 MELAS patients. The A3243G mutation ratio in urine was 62% +/- 9%, significantly higher than that in the blood [(36% +/- 10%), t = -11.13, P < 0.01]. A3243G mutations were detected in at least one tissue of the 28 maternal relatives. The A3243G mutation rates in their urine samples was 33.0% (5.0% - 70.4%), significantly higher than that in their blood samples [8.0% (0 - 33.3%), z = -4.197, P < 0.01]. There was no significant difference in A3243G mutation ratio among the samples of hair follicle, saliva, and blood. CONCLUSION: The A3243G mutation ratio in urine is significantly higher than those in blood samples of the patients and their maternal relatives. A noninvasive method, A3243G mutation ratio analysis of urine is superior to that in blood.

[In vivo interleukin-10 gene transfer down-regulates myocardial matrix metalloproteinase and myocardial collagen expressions in rats with acute myocardial infarction].

OBJECTIVE: We investigated the in vivo effects of recombinant adenovirus-associated virus type-2 (AAV-2) mediated interleukin-10 (IL-10) gene transfer on the expression of matrix metalloproteinase (MMP)-2, 9, tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I and type III in a rat acute myocardial infarction model. METHOD: Male Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 6): sham operation group, MI/AAV2 group, and MI/AAV2-IL-10 group (10(10) vg/ml x 0.1 ml injection at peri-infarct regions immediately post MI). Five days later, the expressions of MMP-2 and MMP-9 were measured by RT-PCR, Western blot and zymography. The expression of TIMP-1 was measured by RT-PCR and Western blot. Collagen type I and type III were assessed by RT-PCR and immunohistochemical stain. RESULTS: The myocardial expressions of MMP-2, MMP-9 and collagen contents in MI/AAV2 group were significantly increased than those in sham operation group. Myocardial expressions of MMP-2, MMP-9 were significantly decreased and the expression of TIMP-1 significantly increased in the MI/AAV2-IL-10 group than those in MI/AAV2 group. Moreover, the expressions of collagen type I, collagen type III and the ratio of I/III collagen in border zones of infarcted myocardium were decreased by 47.6% (P < 0.01), 23.6% (P < 0.05), and 17.9% (P < 0.05) respectively, while the expression of TIMP-1 increased by 73.1%(P < 0.05) in MI/AAV2-IL-10 group compared to MI/AAV2 group. CONCLUSION: In vivo myocardial IL-10 transfer reduced myocardial MMP and collagen expression and increasing the TIMP expression.

Targeted high-throughput sequencing technique for the molecular diagnosis of primary immunodeficiency disorders.

The aim of this study was to investigate the usefulness of targeted high-throughput sequencing (HTS) for the molecular diagnosis of primary immunodeficiency diseases (PID).A total of 56 clinically diagnosed or suspected PID patients were divided into 4 groups according to the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015 and their chief clinical presentations. Patients and their biological family members were examined by targeted HTS, which sequenced the exons and ±10 bp flanking introns of 171 PID-related genes panel. All significant variants were confirmed by PCR-Sanger sequencing. Pathogenicity of the variants was evaluated by using bioinformatics.A total of 117 variants in 73 genes were found in 56 patients. Accurate molecular diagnosis of PID was made in 13 (23.2%) patients, and 12 novel mutations were detected in these patients. Twenty-seven patients carried heterozygous variants that are probably pathogenic in ≥2 genes; 16 patients had only 1 missense variant, or had several variants but not >1 variant was deleterious as evaluated by bioinformatics. The meaning of the targeted HTS results of these patients remains to be studied.Targeted HTS can make a precise molecular diagnosis of PID and detect more novel pathogenic mutations. More and more variations with ambiguous significance are discovered and explanation of these variations is a challenge to the clinicians.

[Molecular genetic analysis of mitochondrial DNA C1494T mutation in non-syndromic hearing loss of Chinese population].

OBJECTIVE: To conduct a molecular epidemiological survey on the mitochondrial DNA C1494T mutation in non-syndromic hearing loss patients in Chinese population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to screen the mitochondrial DNA 12S rRNA C1494T mutation in 20 patients with aminoglycoside antibiotic induced hearing loss, 136 sporadic non-syndromic hearing loss patients and 50 probands of pedigrees with non-syndromic hearing loss. RESULTS: The C1494T mutation did not appear in all cases except for the positive control. CONCLUSION: Incidence of mitochondrial DNA C1494T mutation is much lower than that of mitochondrial DNA A1555G mutation in non-syndromic hearing loss of Chinese population. Mitochondrial DNA C1494T mutation may be a rare variation in non-syndromic hearing loss and is not the main cause of aminoglycoside antibiotic induced-deafness.

[In vitro study about the inhibitory effect of CDAII in combination with sodium butyrate on breast cancer cells].

OBJECTIVE: To investigate if the combined use of CDAII and sodium butyrate can induce demethylation and re-expression of retinoic acid receptor beta2 (RARbeta2) gene in cultured human breast cancer cells MCF7. To explore if the two drugs can inhibit cell growth and induce cell apoptosis synergetically. METHODS: MCF7 cell line was treated with CDAII, sodium butyrate, combination of the two drugs respectively. Methylation was assessed by methylation-specific polymerase chain reaction (MSP) for RARbeta2 gene. Gene expression was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling(TUNEL) and Hoechst33342/propidiumiodide (PI) staining. Cell growth inhibition was measured by MTT assay. RESULTS: Neither CDAII nor sodium butyrate induced demethylation and re-expression of RARbeta2 gene, Combination of the two drugs partially demethylated gene promoter accompanied by re-expression of RARbeta2. The apoptotic cells in the double-drug group were obvious following Hoechst33342/PI staining. The percentage of apoptotic cells in the double-drug group was significantly higher than that of the two single-drug group (39.5% vs 5.2%, 8.1%) (P<0.05). MTT assay showed that compared with single drug,combination of the two drugs inhibited cell growth more significantly, and the two drugs showed interaction (F=15, P<0.05). CONCLUSION: We showed that CDAII, in combination with histone deacetylase inhibitor sodium butyrate synergetically inhibited the proliferation of MCF7 breast cancer cell line and induced apoptosis, in which demethylation and re-expression of RARbeta2 gene induced by combination of the two drugs may be involved.

[Significance of endogenous sulfur dioxide in the regulation of cardiovascular system].

Since the 1980's nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H(2)S), the endogenous gas molecules produced from metabolic pathway, have been realized as signal molecules to be involved in the regulation of body homeostasis and to play important roles under physiological and pathophysiological conditions. The researches on these endogenous gas signal molecules opened a new avenue in life science. To explore the new member of gasotransmitter family, other endogenous gas molecules which have been regarded as metabolic waste up to date, and their biological regulatory effects have been paid close attention to in the current fields of life science and medicine. Sulfur dioxide (SO(2)) can be produced endogenously from normal metabolism of sulfur-containing amino acids. L-cysteine is oxidized via cysteine dioxygenase to L-cysteinesulfinate, and the latter can proceed through transamination by glutamate oxaloacetate transaminase (GOT) to beta-sulfinyl pyruvate which decomposes spontaneously to pyruvate and SO(2). In mammals, activated neutrophils by oxidative stress can convert H(2)S to sulfite through a reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase-dependent process. The authors detected endogenous production of SO(2) in all cardiovascular tissues, including in heart, aorta, pulmonary artery, mesenteric artery, renal artery, tail artery and the plasma SO(2) content. As the key enzyme producing SO(2), GOT mRNA in cardiovascular system was detected and found to be located enriched in endothelial cells and vascular smooth muscle cells near the endothelial layer. When the normal rats were treated with hydroxamate(HDX), a GOT inhibitor, at a dose of 3.7 mg/kg body weight, the blood pressure (BP) went high markedly, the ratio of wall thickness to lumen radius was increased by 18.34%, and smooth muscle cell proliferation was enhanced. The plasma SO(2) level in the rats injected with 125 micromol/kg body weight SO(2) donor was increased to 721.98+/-30.11 micromol/L at the end of 30 seconds, while the blood pressure was decreased to the lowest point 65.0+/- 4.9 mm Hg at the end of 1 minute. The above results showed that endogenous SO(2) might be involved in the maintenance of blood pressure and normal vascular structure. In spontaneous hypertensive rat (SHR) animal model, exogenous supplement of SO(2) donor decreased the BP, the media cross-sectional area, and pressure of the media and the ratio of wall thickness to lumen radius in the SHR. Moreover, the proliferative index of aortic smooth muscle cells was decreased in the SHR treated with SO(2) donor compared with that in SHR. The above data showed that SO(2) could prevent the aortic structural remodeling by inhibiting the proliferation of aortic smooth muscle cells. The authors observed the direct vasorelaxant effects of SO(2) on the aortic ring pre-treated with norepinephrine (NE). SO(2) donor at a concentration of 25-100 micromol/L relaxed the aortic ring temporarily and slightly, but SO(2) donor at a concentration of 1-12 mmol/L induced relaxation of the ring in a concentration-dependent manner. Administration with nicardipine, an L-type calcium channel blocker other than glibenclamide, an ATP sensitive potassium channel (K(ATP) channel) blocker or removal of vascular endothelium could decrease the SO(2)-induced vasorelaxation. In hypoxic pulmonary hypertension animal model, SO(2) donor decreased the mean pulmonary artery pressure and the systolic pulmonary artery pressure (P<0.01), respectively as compared with hypoxic group, and alleviated obviously the hypoxic pulmonary vascular structural remodeling. The percentage of muscularized arteries of small pulmonary vessels was significantly decreased in hypoxia+SO(2) donor-treated rats compared with that of hypoxic rats (P<0.01), while the percentage of non-muscularized vessels was obviously higher in hypoxia with SO(2) donor-treated rats than that of hypoxic rats (P<0.01). Similarly, SO(2) obviously decreased relative media area and relative media thickness of small muscularized pulmonary arteries in hypoxic rats (P<0.01). The above data showed that SO(2) might play an important role in development of hypoxic pulmonary hypertension. Perfusion with SO(2) donor (10(-6)-10(-3) mol/L) to the isolated rat heart obviously inhibited the left ventricular peak rate of contraction ( + LV dp/ dtmax) , peak rate of relaxation (-LV dp/ dtmax) and difference of left ventricular pressure ( DeltaLVP) in a concentration dependent manner. Nicardipine, an L-type calcium channel blocker, could partly antagonize the inhibitory effect of SO(2) on the heart function. In a word, SO(2) could be endogenously generated in cardiovascular tissues and exert important cardiovascular effects such as vasorelaxant effect and negative inotropic effects. Moreover, SO(2) might play considerable roles in the regulation of systemic circulatory pressure, pulmonary circulatory pressure and vascular structural remodeling in the pathogenesis of hypertension and hypoxic pulmonary hypertension. On the basis of the above findings, we presumed that endogenous SO(2) might be a novel cardiovascular functional regulatory gasotransmitter. More studies on the significance of endogenous SO(2) in cardiovascular system under physiological and pathophysiological conditions need to be investigated.

Alteration of vascular urotensin II receptor in mice with apolipoprotein E gene knockout.

Urotensin II (U-II), originally identified as a fish neuropeptide, exerts a broad spectrum of biological functions and is considered to be the most potent vasoconstrictor in mammals. Recently the intense staining of U-II-like immunoreactivity within coronary atheroma and the effect of mitogenic in vascular smooth muscle cells (VSMCs) suggest that U-II is possibly a proatherogenic factor in the pathogenesis of cardiovascular diseases (CVD). In the present study, we analyzed the gene expression of U-II and GPR14, a high-affinity receptor for U-II, in aorta of apolipoprotein E (apoE) -/- mutant mice by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Compared with wild-type mice at the same age group, GPR14 mRNA level is significant increase in aorta of apoE -/- mice at age of 18, 28 and 38 weeks, respectively. The increased GPR14 mRNA level was 54.2, 50.0 and 97.0% in the three age groups, respectively. We did not detect U-II mRNA expression in aorta either in apoE -/- mice or in wild-type mice. In 28-week mice, ligand-binding assay showed that maximum binding capacity (Bmax) of 125I-U-II aorta from apoE -/- mice was increased by 64%, while the affinity of the binding did not change compared with that of wild-type mice. These results indicate that the up-regulation of vascular U-II receptor (UT), GPR14 might be involved in the pathogenesis of atherosclerosis.

Successive alterations of hippocampal gamma-aminobutyric acid B receptor subunits in a rat model of febrile seizure.

Febrile seizure (FS) is a frequently encountered seizure type in childhood. Changes of brain function following FS have clinical importance. The recently identified gamma-aminobutyric acid B receptor (GABA(B)R) is a metabotropic receptor of GABA. In this study, we used a rat model of recurrent FS to investigate the changes of GABA(B)R1a and GABA(B)R2 subunits in hippocampus after recurrent FS by using Western blot, quantitative RT-PCR, double immunofluorescence, in situ hybridization and immunoprecipitation/Western blot. After treatment of hyperthermia and the presence of induced seizures once every 2 days for 10 times, GABA(B)R1a and GABA(B)R2 subunits in hippocampus were decreased after 24 h of the last treatment. The decrease of GABA(B)R1a lasted for 15 days but that of GABA(B)R2 persisted for more than 30 days. The binding of GABA(B)R1a to GABA(B)R2 in hippocampus was also decreased significantly after 24 h of the last treatment and lasted for more than 30 days. In situ hybridization showed that GABA(B)R1a mRNA was significantly decreased in dentate gyrus, and GABA(B)R2 mRNA was considerably reduced in CA3 region. In H10 and FS1 groups in which hyperthermia treatment was the same but no (H10 group) or only one seizure (FS(1) group) was induced, the decrease of GABA(B)R1a and GABA(B)R2 subunits and the reduced binding capability between GABA(B)R1a and GABA(B)R2 subunits were also detected but with less severity, and the time recovering from these abnormalities was shorter. We conclude that GABA(B)R1a and GABA(B)R2 subunits and the binding of the 2 subunits decrease in hippocampus for a relatively long period of time after recurrent FS in immature rats. These changes may result in long-lasting imbalance of excitation/inhibition function in hippocampus, and are derived from the consequences of recurrent febrile seizures.

A novel CFTR mutation found in a Chinese patient with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is rare in Chinese. We investigated the mutations in the gene of cystic fibrosis transmembrane conductance regulator (CFTR) in a Chinese CF patient and reviewed the clinical features, gene mutations in Chinese CF cases. METHODS: Blood samples were collected from a previously reported CF girl and her parents. The 24 coding exons of CFTR of the proband were amplified and sequenced. RESULTS: A Chinese girl of 16 years old was diagnosed as CF at the age of 14. She had recurrent productive cough with bronchiectasis in bilateral upper lobes, parasinusitis and otitis media, but without pancreatic involvement. Her sweat chloride was (108.9 +/- 3.3) mmol/L. A heterozygous novel missense mutation of 699 C --> A which results in the amino acid change of N189K was identified in exon 5. In addition, a heterozygous 3821 - 3823 delT mutation in exon 19 was found in CFTR. The mutation 699C --> A was inherited from her father, and the 3821 - 3823 delT mutation was from her mother. Twenty patients with CF in Chinese reported from 1974 to 2004 were also reviewed. DelF508 mutation was not found in the nine cases whose CFTR mutations were analyzed. CONCLUSIONS: The CF proband carries two heterozygous mutations (699C --> A and 3821 - 3823 delT) in CFTR. 699C --> A mutation is a novel mutation which is not reported previously. Review of reported Chinese cases suggests that the genotype of Chinese CF may be different from those of white cases. More studies are needed to understand the spectra of CFTR and clinical CF features in Chinese.

Febrile seizure, but not hyperthermia alone, induces the expression of heme oxygenase-1 in rat cortex.

BACKGROUND: Febrile seizure (FS) is the most common seizure disorders. Approximately one third of children with a febrile seizure have recurrent events. The mechanism of FS remains unclear. Heme oxygenase-1 (HO-1) is a member of the heat shock proteins family and can be induced in the brain by various stresses, including hyperthemia and seizure. This study aimed at investigating the changes of HO-1 in the cortex of rats after recurrent FS. METHODS: FS in rats was induced ten times, once every 2 days. In a bath of warm water, developing rats were randomly divided into two groups: control group (n = 16) and warm water-treated group (n = 50). The latter group was subdivided into hyperthermia group (n = 19) and FS group (n = 23). The expression and content of HO-1 mRNA in cortex were observed using in situ hybridization and quantitative reverse transcription-polymerase chain reaction (RT-PCR). The content of HO-1 protein in cortex was measured using Western blotting. RESULTS: HO-1 mRNA expression of cortex neurons in FS group was markedly increased in comparison with those in hyperthermia and control groups (P = 0.00), however, there was no statistic difference between hyperthermia group and control group (P = 0.16). The relative amount of HO-1 mRNA in cortex in FS group was increased by 53.13% and 96% in comparison with those in hyperthermia group and control group respectively (P = 0.00), but there was no obvious difference between the later two groups (P = 0.051). Western blotting analysis showed that the HO-1 protein content in cortex in FS group was increased by 198% and 246% in comparison with those in hyperthermia group and control group respectively (P = 0.00). There was no obvious difference in HO-1 protein content between the later two groups (P = 0.09). CONCLUSIONS: Recurrent FS in rats can cause the increase of HO-1 mRNA and protein in cortex which may be involved in the mechanism of FS. The short-time recurrent hyperthermia can not induce the increase of HO-1 mRNA and protein.