Clinical value of biomarkers in diagnosis and treatment of idiopathic pulmonary fibrosis / 南方医科大学学报

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia characterized by progressive accumulation of fibroblastic foci and destruction of the alveolar structure. Due to an incomplete understanding of the mechanism of the occurrence and progression of IPF, currently no effective means have been available for its early screening or treatment. With a poor overall prognosis, the patients with IPF have a median survival of only 2-4 years. In recent years, several studies have confirmed that dozens of molecules are involved in the development of IPF and can be used as potential biomarkers. These biomarkers play important roles in early diagnosis (such as SP-D, MMP-7, and osteopontin), prognostic evaluation (such as telomerase length, KL-6, mtDNA, HSP-70, LOXL2, CXCL13, miRNA, ICAM-1, and CCL18), and guiding treatment of IPF (such as TOLLIP rs3750920 genotype, SAMS score, and SP-D), and also provide potential therapeutic targets (such as TERT, TERR, RTEC, and PARN).

Clinical value of biomarkers in diagnosis and treatment of idiopathic pulmonary fibrosis / 浙江大学学报·医学版

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia characterized by progressive accumulation of fibroblastic foci and destruction of the alveolar structure. Due to an incomplete understanding of the mechanism of the occurrence and progression of IPF, currently no effective means have been available for its early screening or treatment. With a poor overall prognosis, the patients with IPF have a median survival of only 2-4 years. In recent years, several studies have confirmed that dozens of molecules are involved in the development of IPF and can be used as potential biomarkers. These biomarkers play important roles in early diagnosis (such as SP-D, MMP-7, and osteopontin), prognostic evaluation (such as telomerase length, KL-6, mtDNA, HSP-70, LOXL2, CXCL13, miRNA, ICAM-1, and CCL18), and guiding treatment of IPF (such as TOLLIP rs3750920 genotype, SAMS score, and SP-D), and also provide potential therapeutic targets (such as TERT, TERR, RTEC, and PARN).

Progress in genetic research on familiar aneurysms / 中南大学学报(医学版)

The subarachnoid hemorrhage (SAH) caused by ruptured intracranial aneurysms (IAs) is always a lethality. Increasing evidence suggests a familiar aggregation of IA occurrence, which may relate to genetics and there might be an increasing number of IAs in IA families when mutation of disease genes is aggregating. With the progress in the study of familiar intracranial aneurysms (FIAs), a large number of chromosome fragments are found to be related with IAs, such as 1p36, 5q31, 7q11, 14q22, 17cen, 19q13, Xp22. Further studies indicated that mutation of several genes could be the cause of FIAs, including TNFRSF13B, ANRIL, SOX17, ADAMTS15, RNF213 and LOXL2. The independent genetic epidemiologic study on aneurysm families can be used to discover the related genes more effectively, and to explore the mechanism of occurrence of IAs. It's also the precondition for the prevention of disease.

Exfoliation syndrome associated with LOXL1 gene polymorphisms in a Black patient from Latin America: a case report / Síndrome de esfoliação associada a polimorfismos do gene LOXL1 em paciente negro da América Latina: relato de caso

ABSTRACT A 89-year-old Black female with a 6-year history of advanced open-angle glaucoma was referred to the Glaucoma Service of the Ophthalmology Department - Federal University of São Paulo (UNIFESP). Best-corrected visual acuity was 20/400 in the right eye and 20/60 in the left eye. Pseudoexfoliation material was observed at the iris border, angle, and the anterior lens surface. Anterior biomicroscopy revealed exfoliation material forming an evident peripheral zone and a central disc separated by a clear intermediate zone on the anterior lens surface OU. Gonioscopy showed an open-angle Sampaolesis's line and whitish material deposits OU. Fundus examination revealed a cup-to-disc ratio of 1.0 OU with peripapillary atrophy. Genetic analysis for single nucleo­tide polymorphisms of the lysyl oxidase-like 1 gene linked to exfoliation syndrome identified two such single nucleotide polymorphisms, rs1048661 and rs216524.

RESUMO Uma mulher negra de 89 anos com um histórico de seis anos de glaucoma avançado de ângulo aberto avançado foi encaminhada ao Serviço de Glaucoma do Departamento de Oftalmologia da Universidade Federal de São Paulo (UNIFESP). A acuidade visual melhor corrigida era 20/400 no olho direito e 20/60 no olho esquerdo. Material pseudo-exfoliativo foi observado na borda iriana, ângulo e superfície anterior do cristalino. A biomicroscopia de segmento anterior demonstrou material exfoliativo formando uma zona periférica evidente e um disco central separado por uma zona intermediária livre na cápsula anterior do cristalino. A gonioscopia mostrou uma linha de Sampaolesi de ângulo aberto e depósitos esbranquiçados. O exame de fundo de olho revelou disco óptico com escavação total em ambos os olhos com atrofia peripapilar. A análise genética para polimorfismos de nucleotídeo único do gene semelhante à lysyl oxidase-like 1 ligado à síndrome de esfoliação identificou dois desses polimorfismos de nucleotídeo único, rs1048661 e rs216524.

Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus

Background: Glycine oxidase (GO), a type of D-amino acid oxidase, is of biotechnological interest for its potential in several fields. In our previous study, we have characterized a new glycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against all the tested substrate was obtained by screening a random mutant library of BceGO. It is observed that the residue N336 is invariable between its homogeneous enzymes. This work was aimed to explore the role of the residue N336 in glycine oxidase by site-directed mutagenesis, kinetic assay, structure modeling and substrate docking. Results: The results showed that the affinity of N336H, N336K and N336R increased gradually toward all the substrates, with increase in positive charge on side chain, while N336A and N336G have not shown a little significant effect on substrate affinity. The structure modeling studies indicated that the residue Asn336 is located in a random coil between -J-18 and a-10. Also, far-UV CD spectra-analysis showed that the mutations at Asn336 do not affect the secondary structure of enzyme. Conclusion: Asn336 site was located in a conserved GHYRNG loop which adjoining to substrate and the isoalloxazine ring of FAD, and involved in the substrate affinity of glycine oxidase. This might provide new insight into the structure-function relationship of GO, and valuable clue to redesign its substrate specificity for some biotechnological application.

Association between LOXL1 gene polymorphisms and primary open angle glaucoma in Sichuan population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate association between the lysyl oxidase-like 1 (LOXL1) gene single nucleotide polymorphism (SNP) and primary open-angle glaucoma (POAG) in Sichuan population.</p><p><b>METHODS</b>In this study,416 subjects with primary open-angle glaucoma and 997 normal controls were recruited.Three reported LOXL1 tag SNPs (rs1048661,rs3825942 and rs2165241) were genotyped by SNaPshot method.</p><p><b>RESULTS</b>The study showed that the genotypes of LOXL1 rs1048661,rs3825942 and rs2165241 between POAG and control groups were not statistically significant (OR=1.085, 95%CI 0.92-1.28, P=0.578 for rs1048661; OR=1.059, 95%CI 0.82-1.37, P=0.846 for rs3825942; OR=1.006, 95%CI 0.77-1.32, P=0.966 for rs2165241, respectively). There were no significant difference in allele frequency distribution of LOXL1 rs1048661、rs3825942 and rs2165241 between POAG and normal controls (P=0.322, P=0.660, P=0.965).</p><p><b>CONCLUSION</b>The results from the present study do not indicate the association of LOXL1 SNPs (rs1048661, rs3825942 and rs2165241) with POAG in Sichuan population.</p>

Effect of residue Y76 on co-enzyme specificity of meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum / 生物工程学报

In industrial application of NAD(P)H-dependent dehydrogenases, NAD(H) has the advantages over NADP(H) in higher stability, lower price and wider recycling system. Recently, a meso-2,6-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH) has been found to be a useful biocatalyst for the production of D-amino acids, but it requires NADP(H) as co-enzyme. To switch the co-enzyme specificity from NADP(H) to NAD(H), we studied the effect of Y76 on the co-enzyme specificity of StDAPDH, because the crystal structural analysis indicated that residue Y76 is near the adenine ring. The mutation of Y76 exerted significant effect on the co-enzyme specificity. Furthermore, the double mutant R35S/R36V significantly lowered the specific activity toward NADP+, and the combination of R35S/R36V with some of the Y76 mutants resulted in mutant enzymes favorable NAD+ over NADP+. This study should provide useful guidance for the further development of highly active NAD(+)-dependent StDAPDH by enzyme engineering.

Enzymatic production of α-ketoglutaric acid by L-glutamate oxidase from L-glutamic acid / 生物工程学报

We produced α-ketoglutaric acid (α-KG) from L-glutamic acid, using enzymatic transformation approach with L-glutamate oxidase (LGOX). First, wild strain Streptomyces sp. FMME066 was mutated with NTG, a genetically stable mutant Streptomyces sp. FMME067 was obtained. Under the optimal nutrition conditions with fructose 10 g/L, peptone 7.5 g/L, KH2PO4 1 g/L and CaCl2 0.05 g/L, the maximum LGOX activity reached 0.14 U/mL. The LGOX was stable to pH and temperature, and Mn2+ had a stimulating effect. Finally, after 24 h enzymatic conversion under the optimal conditions, the maximum titer of α-KG reached 38.1 g/L from 47 g/L L-glutamic acid. Enzymatic transformation by LGOX is a potential approach for α-KG production.

Identification of the involvement of LOXL4 in generation of keratocystic odontogenic tumors by RNA-Seq analysis / 国际口腔科学杂志·英文版

Keratocystic odontogenic tumors (KCOT) are benign, locally aggressive intraosseous tumors of odontogenic origin. KCOT have a higher stromal microvessel density (MVD) than dentigerous cysts (DC) and normal oral mucosa. To identify genes in the stroma of KCOT involved in tumor development and progression, RNA sequencing (RNA-Seq) was performed using samples from KCOT and primary stromal fibroblasts isolated from gingival tissues. Seven candidate genes that possess a function potentially related to KCOT progression were selected and their expression levels were confirmed by quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay. Expression of lysyl oxidase-like 4 (LOXL4), the only candidate gene that encodes a secreted protein, was enhanced at both the mRNA and protein levels in KCOT stromal tissues and primary KCOT stromal fibroblasts compared to control tissues and primary fibroblasts (P<0.05). In vitro, high expression of LOXL4 could enhance proliferation and migration of the human umbilical vein endothelial cells (HUVECs). There was a significant, positive correlation between LOXL4 protein expression and MVD in stroma of KCOT and control tissues (r=0.882). These data suggest that abnormal expression of LOXL4 of KCOT may enhance angiogenesis in KCOT, which may help to promote the locally aggressive biological behavior of KCOT.

Immunohistochemical localization of aipha-fetoprotein in liver of adult male albino rats treated with homocysteine and trimethylglycine

Alpha-fetoprotein[AFP] has been reported to be associated with hepatocellular carcinoma. Homocysteine [HCY] could be one of the factors contributing in elevating alpha-fetoprotein level in the rat liver. We aimed at investigating the effect of HCYon rat liver and studying the protective effect of Trimethyiglycine [TMG]. Adult albino rats were made hyperhomocystenamic by oral supplementation of 0.6mg/kg homocysteine compared to control and trimethylglycine 6gmlkg treated groups for 6 weeks of experimental duration. Liver was prepared for histopathological, histochemical studies. AFP was investigated by immunohistochemical techniques. Higher reaction of aipha-fetoprotein was observed in rat liver of HCY treated group rather than the interacting group, compared to-ye reaction in both control and TMG treated groups. Histopathological results of HCY treated rats revealed liver hepatocytes with cytoplasmic vacuolation and pyknotic nuclei. Dilatation of the central viens with interstitial haemorrhage and amyloid deposition were also observed. Kupfer cells and lymphocytes were both apparently increased. These changes were improved markedly with TMG administration. Histochemical localization for carbohydrates showed decrease reaction in hepatocytes of HCY treated group rather than other groups. It is concluded that HCY induced severe hepatic alterations. These alterations, were less prominent in animals treated with TMG indicating that TMG can be possible hepatoprotector against HCYinduced toxicity

Characterization of attenuated Salmonella C500 strain with a delta asd mutant and use as an Asd+ balanced-lethal host-vector system / 生物工程学报

Salmonella enterica serovar Choleraesuis strain C500 is a live, attenuated vaccine that has been used in China for over 40 years to prevent piglet paratyphoid. The objective of this study was to evaluate the potential of attenuated Salmonella enterica serovar Choleraesuis C500 strain with a delta asd mutant as an effective live vaccine vector by the Asd+ balanced-lethal host-vector system. Here, we compared the characteristics of S. enterica serovar Choleraesuis delta asdC500 strain with the parent C500 strain, including phenotype, growth rate, virulence, safety, and expression for heterologous antigen. The mean generation times of delta asdC500 mutant, the vector control delta asdC500 (pYA3493), and the parent avirulent C500 vaccine strain in Luria broth were 30.7, 28.1, and 27.9 min, respectively. The fermentation patterns of theses three strains on different carbohydrates, and the levels of production of H2S, were similar. The O and H antigens of delta asdC500 mutant, delta asdC500 (pYA3493) and delta asdC500 (pYA-F1P2) were 6,7:C:1,5, identical to the parent strain C500. By the method of Reed and Muench, groups of mice were challenged by the intraperitoneal route with different amounts of delta asdC500 (pYA3493) or the parent C500 strain, and the virulence of delta asdC500 (pYA3493) with LD50 of 1.1 x 10(7) CFU was a little lower than C500 with LD50 of 4.4 x 10(6) CFU. All piglets inoculated with delta asdC500 (pYA3493) or C500 survived, and no signs of disease were observed during the entire experimental period. No major differences were found in these two groups. In addition, the recombinant pYA-F1P2 plasmid was very stable in the recombinant delta asdC500 (pYA-F1P2) strain, which expressed secretorily a large amount of the recombinant filamentous hemagglutinin type I domain and pertactin region 2 domain antigen (rF1P2) of Bordetella bronchiseptica. In this study, we have shown that the delta asdC500 mutant had a series of biological characteristics similar to the parent vaccine strain C500. Furthermore, the strain could express secretorily a large amount of heterologous antigen. It is likely that this Salmonella expression and delivery system could be easily adapted to develop multivalent recombinant Salmonella vaccines against infectious agents using the Asd+ balanced-lethal host-vector system.

Development of biosensor based on immobilized L-glutamate oxidase for determination of monosodium glutamate in food.

A monosodium glutamate (MSG) biosensor with immobilized L-glutamate oxidase (L-GLOD) has been developed and studied for analysis of MSG in sauces, soup etc. The immobilized enzymatic membrane was attached with oxygen electrode with a push cap system. The detection limit of the sensor was 1 mg/dl and the standard curve was found to be linear upto 20 mg/dl. Response time of the sensor was 2 min. Cross-linking with glutaraldehyde in presence of Bovine Serum Albumin (BSA) as a spacer molecule has been used for immobilization. Optimization of the sensor was done with an increase in L-GLOD concentration (6.3-31.5 IU) and also with increase in loading volume of enzyme solution (5-20 microl). Optimization of pH and temperature was also studied. The permeability of O2 through different membrane was studied with and without immobilized L-GLOD. The enzymatic membrane was used for over 20 measurements and stability of the membrane was observed.

Construction and characterization of delta crp delta asd mutant host-vector balanced lethal system of Salmonella choleraesuis C500 strain / 生物工程学报

Salmonella choleraesuis C500 strain was an attenuated vaccine strain to prevent piglet paratyphoid, attenuated by chemical method. Although the vaccine has good immunogenicity, it remains some residual virulence. In order to develop a safer vaccine strain and exploit C500 as a live vaccine vector for mucosal immunization, delta crp delta asd double deletion mutant was constructed. Firstly, the recombination suicide vector with 320 bp-deleted crp (cAMP receptor protein) gene and sacB (sucrose-sensitive gene) gene was constructed and conjugated with C500. The unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR. Then the asd (beta-aspartic semialdehyde dehydrogenase) gene was further deleted in the delta crp strain by the same method. Foreign DAP (diaminopimelic acid) must be supplied for delta crp delta asd mutant to grow. The phenotype, growth properties and virulence in mice of delta crp mutant were further characterized. In conclusion, the delta crp delta asd double-deletion mutant was successfully constructed. The delta crp delta asd mutant can be used as a live vector to express foreign genes and to develop potential oral multivalent vaccines.

Isolation, purification, and characterization of L-glutamate oxidase from Streptomyces sp. 18G

An extracellular L-glutamate oxidase (GLOD) was purified from soil-isolated Streptomyces sp 18G. The enzyme had a molecular weight of approximately 120,000 and consisted of two identical subunits, each with a molecular weight of 61,000. The isoelectric point was pH 8.5 and the enzyme had an optimal pH between 7.0-7.4. GLOD showed the maximum activity at 37ºC. The GLOD activity was stable at pH ranging from 6.5 to 7.0 for 1 hr. Among 21 amino acids tested for substrate specificity, L-glutamate was almost exclusively oxidized. D-glutamate and L-aspartate were oxidized but only to extents of 0.79 percent and 0.53 percent, respectively.

Integration of different T-DNA structures of ACC oxidase gene into carnation genome extended cut flower vase-life differently / 生物工程学报

The cultivar 'Master' of carnation (Dianthus caryophyllus L.) was transformed with four T-DNA structures containing sense, antisense, sense direct repeat and antisense direct repeat gene of ACC oxidase mediated by Agrobacterium tumefaciens. Southern blotting detection showed that foreign gene was integrated into the carnation genome and 14 transgenic lines were obtained. The transgenic plants were transplanted to soil and grew normally in greenhouse. Of the 12 transgenic lines screened, the cut flower vase life of 8 transgenic lines is up to 11 days and the longest one is 12.8 days while the vase life of the control is 5.8 days under 25 degrees C. The vase life of 2 lines out of 3 with single sense ACO gene is same as that of the control, while the vase life of 3 lines out of 4 with single antisense ACO gene is prolonged. The vase life of cut flowers of 5 lines with direct repeat ACO genes is all prolonged by about 6 days, while the vase life of 3 out of 7 lines with single ACO gene is same as that of the control. During the senescence of cut flowers, the ethylene production of the most of the transgenic lines decreased significantly, and the production of ethylene is not detectable in lines T456, T556 and T575. The results of the research demonstrate that antisense foreign gene inhibits expression of endogenesis gene more significantly than sense one. Both sense direct repeat and antisense direct repeat foreign genes can suppress endogenous gene expression more significantly comparing to single foreign genes. The transgenic lines obtained from this research are useful to minimize carnation cut flower transportation and storage expenses.

Progress of studies on VEC apoptosis-inducing proteins in snake venom and its mechanism--review / 中国实验血液学杂志

Hemorrhagic snake venom specially induces apoptosis of VEC (vascular endothelial cells). Five apoptosis-inducing proteins had been purified and characterized from crude snake venom. Two of these are L-amino acid oxidase (LAO), the others belong to metalloprotease/disintegrin family. LAO catalyzes H2O2 production by oxidizing some plasma membrane proteins of VEC, disintegrins interfere with binding of integrins with their ligands. The expression of p53 and bcl-2 increases during VEC apoptosis induced by snake venom, moreover, the mRNA of bcl-2 is spliced into two fragments. It has been proved that one of adhesion-dependent signal molecules, alphavbeta3, and one of phospholipid signal molecules, PC-PLC (phosphatidylcholine-specific phospholipase C), are involved in above apoptosis-inducing signal transudation pathway. These results throw light on finding out specific component from protein is snake venom. This component is able to induce tumor vascular endothelial cells apoptosis. This review summarized progress of research on hemorrhagic snake venoms.

Polymorphisms of octopine dehydrogenase (Odh) in mollusks and implications for the neutralism-selectionism hypothesis

Octopine dehydrogenase (Odh) was examined in several species of bivalves and gastropods and complemented with bibliographic data, to assess the controversy between neutralism and selectionism in explaining the maintenance of genetic variation in natural populations. This debate was the center of the molecular evolution and population genetic research in the 1970s and 1980s, but waned thereafter, without resolution. Although DNA data have been produced, implications are not understood. We examined the polymorphims of Odh in several species of bivalves and gastropods, and the kinetic properties (apparent Km) of the different isozymes in the scallop Euvola ziczac that indicates an apparent case of overdominance of the heterozygous individuals. The question "which of the two hypothesis is correct" has shifted with time to "how much influence did each factor have in the maintenance of genetic variation".

Enzymes that regulate ethylene levels--1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, ACC synthase and ACC oxidase.

The plant enzymes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase, catalyze essential steps in the biosynthesis of the phytohormone ethylene; the microbial enzyme ACC deaminase catalyses the hydrolytic cleavage of ACC, the immediate precursor of ethylene, and is therefore an inhibitor of ethylene biosynthesis. In this manuscript, the biochemical properties and mechanisms of these three enzymes and the genes that encode them are examined and compared. Despite the fact that ACC oxidase and ACC deaminase both act on the same substrate, i.e., ACC, these two enzymes and the mechanisms that they employ are quite different. Conversely, although ACC synthase catalyses the synthesis of ACC and ACC deaminase catalyses its hydrolysis, these enzymes share a number of important physical and biochemical properties.

Biochemical characterization of NADPH diaphorase in the chick embryo retina: stimulation by calcium ions and inhibition by arginine analogs

Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is prsent in specific groups of cells in several neural tissues, including the retina. We determined NADPH-diaphorase activity in homogenates of the chick embryo retina. The enzyme activity was measured spectophotometrically at 585 nm after incubating retinal total homogenates (100-150 µg protein) with 1mMNADPH and 0.5 mM nitroblue tetrazolium in 50 mMTris buffer, pH8.1, at 37ºC. NADPH-diaphorse was detected in 14-day old retinas and 53-65 per cent of the enzyme activity was inhibited by 3 mM NG-nitro-L-arginine (NARG), the arginine analog. One mM L-N5-(1-iminoethyl)ornithine (NIO) was the most potent inhibitor (63 per cent inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33 per cent inhibition) and I mMNG-monomethyl-L-arginine acetate (NMMA) (14 per cent inhbition) were less effective. Enzyme activity was increased by 48 per cent by 2 mM calcium chloride, and effect reversed by 1 mMEGTA or EDTA. Basal enzyme levels were also partially inhibited by the chelators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show that the NADPH-diaphorase assay is sample and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated

Negative inotropic effect of carbachol on rat atria mediated by nitric oxide

En este trabajo nosotros mostramos que la aurícula aislada de rata sintetiza óxido nítrico (ON) y este actúa como un mensajero intracelular incrementando la producción de GMPc, que a su vez modula el efecto contráctil inhibitorio ejercido por la activación muscarínica. El carbacol activando al receptor muscrínico M2 estimula el ciclo de los fosfoinosítidos y a la óxido nítrico sintaza con la consiguiente producción de ON. Los inhibidores de fosfolipasa C, proteína quinasa C, calcio/calmodulina, óxido nítrico sintaza y guanilato ciclasa, desvían hacia la derecha la curva dosis-respuesta del carbacol sobre la contractilidad. Más aun, en nitroprusiato de sodio y el 8-bromo GMPc indujeron un efecto inotrópico negativo, similar a las bajas concentraciones de carbacol. Estos reultados sugieren que el carbacol activando a receptores muscarínicos de tipo M2 ejerce un efecto inotrópico negativo asociado a un incremento en la producción de ON. Este mecanismo parece ser secundario a la estimulación del ciclo de los fosfoinosítidos vía la activación de la fosfolipas C; que desencadeando reacciones en cascada llevan a la producción de ON, el cual contribuye al efecto inotrópico negativo ejercido por bajas concentraciones de carbacol