A membrane-associated metalloprotease of Schistosoma japonicum structurally related to the FACE-1/Ste24p protease family.

The CaaX proteases are closely related in the post-translational modification of many membrane-bound or secreted proteins and play a key role in the activation or stabilization of these molecules belonging to the CAAX family. In this study, a full-length cDNA putatively encoding a FACE-1/Ste24p CaaX protease (type I) of the Schistosoma japonicum was isolated. The cDNA, named SjSte24p, composed of 1646 bp and encoded 473 amino acids with predicted Mr/pI as 54.77 kDa/8.04. SjSte24p is a monoexonic gene constantly expressed in the parasite from cercariae to adult stages. It contained the characteristic of CaaX protease topology, including seven trans-membrane domains and a metallo-protease segment with a zinc-binding motif (HEXXH). SjSte24p shared a considerable degree of sequence identity with the type I CaaX proteases. A phylogenetic analysis showed that this protein family is tightly conserved from fungi to vertebrates. The expressed recombinant SjSte24p protein showed a proteolytic activity, which was inhibited by EDTA. Its activity was increased at low doses of the Zn2+ (0.001-0.01 mM); but was reversibly down-regulated at high doses (>0.1 mM). The native SjSte24p appeared to function in insoluble from. The protein was mainly localized in the tegument on the surface of adult worms. These results indicated that the SjSte24p is a practical zinc-dependent metalloprotease, which belongs to the FACE-1/Ste24p protease family.

Isolation and characterization of a novel serine protease inhibitor, SjSPI, from Schistosoma japonicum.

Serine proteinase inhibitor (Serpin, SPI) is a vital superfamily of endogenous inhibitors that monitor proteolytic events active in a number of biological functions. In this study, we isolated a full length gene encoding a novel serine protease inhibitor of Schistosoma japonicum (SjSPI) and characterized its molecular properties. Our result showed that SjSPI contained an open reading frame of 1,218 bp, which encoded 405 amino acid residues. Chromosomal structure analysis showed that SjSPI gene was comprised of six exons separated by five introns. It had essential structural motifs which were well conserved among the Serpin superfamily and showed 17-33% sequence identities with Serpins from other helminthic parasites. Trematode Serpin diverged separately into two different subclades and that the SjSPI clustered Subclade I. Exon-intron structures of trematode Serpins were highly conserved, closely with cestode Serpins. No signal peptide but a strongly transmembrane domain was predicted to exist in SjSPI, suggesting that the protein might be a soluble membrane-associated protein. Homology modeling predicted in silico confirmed that the SjSPI structure also belonged to the Serpin superfamily, containing nine α-helices and a reactive central loop. The bacterially expressed recombinant GST-SjSPI protein effectively inhibited the activities of chymotrypsin, trypsin and thrombin. Expression of SjSPI was detected throughout various developmental stages of the parasite in host and reached its maximal levels at the adult and egg stages, which suggests that SjSPI may be possibly involved in maintaining the physiology of eggs by regulating endogenous serine proteases.

Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis.

BACKGROUND: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite's evasion from the host's immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis. METHODS: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis. RESULTS: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity. CONCLUSIONS: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

Molecular cloning and characterization of a phospholipid hydroperoxide glutathione peroxidase gene from a blood fluke Schistosoma japonicum.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and plays critical roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. A full-length cDNA sequence corresponding to GPx gene from Schistosoma japonicum (designated SjGPx) was isolated and characterized. SjGPx contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3'-untranslated region. Protein encoded by SjGPx demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Phylogenetic analysis revealed that the SjGPx was highly related to the other PHGPx-related members, and clustered into the trematode subclade II. Semi-quantitative reverse transcription PCR and western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference was employed to investigate the effects of knockdown of SjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. We observed a 53.86% reduction in total GPx activity and the eggs severely deformed. Oxidative stimulation of viable worms with H2O2 or paraquat resulted in 1.6- to 2.1-fold induction of the GPx activity. Our results revealed that the SjGPx protein is selenium-dependent PHGPx, which might actively participate in the detoxification of oxidative damage during egg production.

Expression pattern and substrate specificity of Clonorchis sinensis tyrosinases.

Tyrosinase (TYR) is a copper-containing glycoenzyme that mediates hydroxylation of tyrosine into dihydroxyphenylalanine and oxidation of dihydroxyphenylalanine into dihydroxyphenylalanine quinone. TYRs play pivotal roles in eggshell sclerotisation of trematode parasites, while their comprehensive biochemical properties remain elusive. We characterised genes encoding four TYRs (CsTYR1-4) of Clonorchis sinensis, a causative agent of human hepatobiliary disease. These genes shared tightly conserved amino acid residues, two copper binding catalytic motifs and a cysteine-rich epidermal growth factor-like domain. The native and recombinant CsTYRs showed high reactivity against diphenol compounds, especially those with hydroxyl groups in ortho-positions (catechol and l-dihydroxyphenylalanine), but showed minimal activity toward monophenol compounds. Diphenolase activity was enhanced by increased pH of the reaction buffer from 5.0 to 7.0. The temporal induction of CsTYR expression coordinated with the sexual maturation of the worm; enzyme activity was mainly in the vitelline glands and intrauterine immature eggs proximal to the ovary. The primary structures and functional domains of CsTYRs showed significant similarities to those of the vertebrate orthologs, whereas the amino acids shared with the nematode and insect proteins were largely restricted in the bicopper active center. Unlike highly diverged TYR homologs in vertebrates, multiple paralogs have not yet evolved into the separate lineages in trematode genomes, suggesting that duplication of TYR genes might relate to increased genic dosage/redundancy in trematodes. In vitro treatment of copper chelator, diethyldithiocarbamic acid, inhibited generation of phenotypically normal egg. TYR proteins are essential for C. sinensis reproduction, thus might be targeted for therapeutic and vaccine strategies against clonorchiasis, which is prevalent in several Asian countries and is one of the most important predisposing factors for human cholangiocarcinoma. The close phylogenetic relationships between trematode and vertebrate homologs also provide a molecular clue to understand the multifaceted evolutionary pathway of TYR homologs across animal taxa.

Differential activation of diverse glutathione transferases of Clonorchis sinensis in response to the host bile and oxidative stressors.

BACKGROUND: Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. METHODOLOGY/PRINCIPAL FINDINGS: We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular trematodes. Interestingly, secretion of a 28 kDa σ-class GST (Cs28σGST3) was significantly affected by the host bile, involving reduced secretion of the 28 kDa species and augmented secretion of Cs28σGST3-related high-molecular-weight 85 kDa protein. Oxidative stressors induced upregulated secretion of 28 kDa Cs28σGST3, but not an 85 kDa species. A secretory 26 kDa µ-class GST (Cs26µGST2) was increased upon treatment with oxidative stressors and bile juice, while another 28 kDa σ-class GST (Cs28σGST1) showed negligible responses. CONCLUSIONS/SIGNIFICANCE: Our results represent the first analysis of the genuine nature of the C. sinensis ESP proteome in the presence of host bile mimicking the natural host environments. The behavioral patterns of migration and maturation of C. sinensis in the bile ducts might contribute to the secretion of copious amounts of diverse GSTs, but a smaller quantity and fewer kinds of cysteine proteases. The Cs28σGST1 and its paralog(s) detoxify endogenous oxidative molecules, while Cs28σGST3 and Cs26µGST2 conjugate xenobiotics/hydrophobic substances in the extracellular environments, which imply that diverse C. sinensis GSTs might have evolved for each of the multiple specialized functions.

[Construction and transplantation of tissue-engineered skin with mouse embryonic fibroblasts in SD mice].

OBJECTIVE: To investigate the application and mechanism of tissue-engineered skin with mouse embryonic fibroblasts (MEFs) for the full-thickness skin defects on mice. METHODS: The MEFs and fibroblasts were cultured and seeded in scaffold made of rat tail collage. ELISA method was used for detection of secretory function. The full-thickness skin defects were created on mice and covered by MEFs-scaffold complex (experimental group), or FBs-scaffold complex (control group 1), or scaffold only (control group 2). The process of wound healing was evaluated by observation of the re-epithelization rate. Microvessel density (MVD) and vimentin within the wound sites were also detected with immunohistochemistry staining technique to describe the characteristics of wound healing. Hoechst 33342 staining was performed to trace MEFs'fate. RESULTS: MEFs scaffold group had higher level secretion of IL-6 and lower of TGF-beta1 than FBs scaffold group (P<0.05). Compared with wounds in control groups, the wounds in MEFs group healed markedly fast (P<0.05) and the MVD was significantly higher (P <0.05). The fibroblasts in the wounds of MEFs group were arranged regularly and the MEFs decreased during the healing process. CONCLUSIONS: The MEFs-scaffold complex can promote wound healing with less scar formation. MEFs may have an inducing effect on the wound healing.

Polymer-controlled crystallization of unique mineral superstructures.

The origin of complex superstructures of biomaterials in biological systems and the amazing self-assembly mechanisms of their emergence have attracted a great deal of attention recently. Mimicking nature, diverse kinds of hydrophilic polymers with different functionalities and organic insoluble matrices have been designed for the morphogenesis of inorganic crystals. In this Research News, emerging new strategies for morphogenesis and controlled crystal growth of minerals, that is, selective adsorption and mesoscale transformation for highly ordered superstructures, the combination of a synthetic hydrophilic polymer with an insoluble matrix, a substrate, or the air/solution interface, and controlled crystallization in a mixed solvent are highlighted. It is shown that these new strategies can be even further extended to morphogenesis and controlled crystallization of diverse inorganic or inorganic-organic hybrid materials with structural complexity, structural specialties, and improved functionalities.

Modular evolution of glutathione peroxidase genes in association with different biochemical properties of their encoded proteins in invertebrate animals.

BACKGROUND: Phospholipid hydroperoxide glutathione peroxidases (PHGPx), the most abundant isoforms of GPx families, interfere directly with hydroperoxidation of lipids. Biochemical properties of these proteins vary along with their donor organisms, which has complicated the phylogenetic classification of diverse PHGPx-like proteins. Despite efforts for comprehensive analyses, the evolutionary aspects of GPx genes in invertebrates remain largely unknown. RESULTS: We isolated GPx homologs via in silico screening of genomic and/or expressed sequence tag databases of eukaryotic organisms including protostomian species. Genes showing strong similarity to the mammalian PHGPx genes were commonly found in all genomes examined. GPx3- and GPx7-like genes were additionally detected from nematodes and platyhelminths, respectively. The overall distribution of the PHGPx-like proteins with different biochemical properties was biased across taxa; selenium- and glutathione (GSH)-dependent proteins were exclusively detected in platyhelminth and deuterostomian species, whereas selenium-independent and thioredoxin (Trx)-dependent enzymes were isolated in the other taxa. In comparison of genomic organization, the GSH-dependent PHGPx genes showed a conserved architectural pattern, while their Trx-dependent counterparts displayed complex exon-intron structures. A codon for the resolving Cys engaged in reductant binding was found to be substituted in a series of genes. Selection pressure to maintain the selenocysteine codon in GSH-dependent genes also appeared to be relaxed during their evolution. With the dichotomized fashion in genomic organizations, a highly polytomic topology of their phylogenetic trees implied that the GPx genes have multiple evolutionary intermediate forms. CONCLUSION: Comparative analysis of invertebrate GPx genes provides informative evidence to support the modular pathways of GPx evolution, which have been accompanied with sporadic expansion/deletion and exon-intron remodeling. The differentiated enzymatic properties might be acquired by the evolutionary relaxation of selection pressure and/or biochemical adaptation to the acting environments. Our present study would be beneficial to get detailed insights into the complex GPx evolution, and to understand the molecular basis of the specialized physiological implications of this antioxidant system in their respective donor organisms.

Vitellocyte-specific expression of phospholipid hydroperoxide glutathione peroxidases in Clonorchis sinensis.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and may protect against lipid hydroperoxidation in biomembranes. We isolated full-length cDNA sequences encoding four different PHGPxs from a causative agent of cholangiocarcinoma, Clonorchis sinensis (CsGPx1, CsGPx2, CsGPx3 and CsGPx4). These sequences contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in their 3'-untranslated regions. The open reading frames were composed of six exons in the chromosomal segments of CsGPx1 (7705bp), CsGPx2 (5871bp) and CsGPx3 (3867bp) and five exons in CsGPx4 (5655bp). The positions of these introns were tightly conserved between the trematode and vertebrate PHGPx genes. Oxidative stimulation of viable worms with H(2)O(2) or paraquat resulted in 1.5- to 2-fold induction of the GPx activity. The CsGPx proteins were specifically localised in vitellocytes within vitelline follicles and premature eggs in the proximal uterus. In the eggs, glutathione, an electron donor for GPx, was co-localised with the CsGPx proteins, while thioredoxin, which is preferred by peroxiredoxin, was principally detected in the extracellular space between the embryonic cell mass and an eggshell. Our data may suggest a concerted or a specialised function between a thioredoxin-dependent enzyme(s) and GPx in protecting against H(2)O(2)-derived damage during maturation of the embryo and formation of the eggshell, in these catalase-lacking trematode parasites. The uniquely conserved genomic organisation and Sec-dependency amongst trematode and vertebrate PHGPx homologues will also provide insight into the evolutionary episode and functional/biochemical diversification of GPx proteins.

[Research progress on the antioxidant enzyme family in medical trematode].

Reactive oxygen species (ROS), generated in the metabolism process of aerobic organisms, can induce oxidative damages in the body. These organisms are all equipped with an excellent defense system to protect themselves and antioxidant enzymes play an important role in the system. Parasitic trematodes have to eliminate ROS not only from themselves but also from the immune system of their hosts. To better understand the structures and specialties of the antioxidant enzymes in trematodes is conducive to the study on reproductive physiology of trematode and on drug and vaccine development. This paper summarizes the research progress on the family of antioxidant enzymes in trematodes including glutathione peroxidase (GPx), superoxide dismutase (SOD) and peroxiredoxin (PRx) in the past decades.

Functional identification of a protein inhibitor of neuronal nitric oxide synthase of Taenia solium metacestode.

The protein inhibitor of neuronal nitric oxide synthase (PIN) performs critical functions in several biological processes including inhibition of neuronal nitric oxide synthase (nNOS) activity, intracellular trafficking of proteins and cellular maturation. In this study, we isolated a gene that putatively encoded a PIN homologue in the Taenia solium metacestode (TsM), a causative agent for neurocysticercosis (NC). A full-length cDNA of 452-bp in length, designated TsMPIN, was found to encode an open reading frame (ORF) of 103 amino acids with a predicted molecular weight of 11.3kDa. This single copy gene possessed an intervening short intron (74bp-long) within its ORF region. The deduced amino acid sequence revealed a substantial degree of sequence identity with the PINs and the dynein light-chains isolated from other organisms (63-81%). TsMPIN ectopically expressed in neuroblastoma N1E115 cells effectively inhibited dimerization of nNOS upon stimulation. The recombinant TsMPIN also negatively regulated the dimerization of recombinant nNOS, which was attenuated significantly by the TsMPIN-specific antibody. TsMPIN was primarily localized in the lining cells of the trabecules and the muscles surrounding the scolex, and was sparsely within the cytosol of the bladder wall. We also identified TsM nNOS-immunoreactive protein by both NADPH-diaphorase histochemical staining, and immunohistochemical localization and immunoprecipitation with antibodies specific to nNOS N-terminus. These two functionally related proteins showed a co-localized expression pattern. Our results strongly suggest that the production of NO in the TsM might be tightly regulated through the nNOS-TsMPIN feedback system to maintain physiological homeostasis in the parasite.

Differential expression of Paragonimus westermani eggshell proteins during the developmental stages.

Eggs of trematode parasites are comprised of numerous vitelline cells and one fertilized ovum, and are encapsulated within a protein shell provided by the vitellocytes. In this study, we isolated two full-length cDNA clones that showed substantial levels of sequence identity with trematode-specific eggshell precursor proteins from the human lung fluke, Paragonimus westermani. These cDNAs, designated Pw-Vit20 (868-bp-long) and Pw-Vit36 (883-bp-long), shared a 76% identity with one another at the nucleotide level, and each encoded a 261-amino acid (aa) polypeptide. The deduced aa sequences contained a N-terminal hydrophobic segment, as well as a sequence motif of Gly-Gly-Gly-Tyr-Asp-Asn/Thr-Tyr-Gly-Lys/Gln, which is highly homologous with the eggshell proteins of Fasciola hepatica. With the high frequencies of tyrosine, glycine and lysine, the positions occupied by tyrosine, which has been proved to be converted into dihydroxyphenylalanine, were well preserved. Pw-Vit20 and Pw-Vit36 were found to be monoexonic genes with variably diverged variants scattered into multiple genomic loci. Their protein products were localized in the vitelline follicles and eggshells. Expression of Pw-Vit20 was restricted to the egg and adult stages, thus suggesting a critical involvement of Pw-Vit20 in the parasite's fecundity activity. Conversely, Pw-Vit36 was constitutively expressed in the metacercariae and juvenile stages in the vitelline follicles and ducts, which suggested that the prepositioning of stem or primordial vitelline cells within the juveniles prior to sexual maturation. Pw-Vit36 might acquire a unique or additional function relevant to the maturation and/or development of the vitelline cells/follicles during the evolutionary period of P. westermani. Differential biological implications of multiple eggshell precursor proteins may provide insight into the molecular mechanism of eggshell formation and the developmental process of the vitelline follicles in the parasitic trematode.

A membrane-associated metalloprotease of Taenia solium metacestode structurally related to the FACE-1/Ste24p protease family.

The CaaX proteases are intimately involved in the post-translational modification of prenylated proteins and play a critical role in the activation/stabilization of membrane-bound or secreted molecules constituting the CAAX protein family. In this study, we have isolated a full-length cDNA putatively encoding a type I CaaX protease of the Taenia solium metacestode (TsM), which an agent causative of human neurocysticercosis. The cDNA, designated TsSte24p, comprised 1,505 bp and coded for an open reading frame of 472 amino acids with predicted Mr 54.5 kDa. This monoexonic TsSte24p gene existed as a single copy within the TsM genome and constantly expressed in the parasite from metacestode to adult stages. The TsSte24p exhibited the typical CaaX protease topology, including seven transmembrane domains and a metalloprotease segment with a zinc-binding motif. It shared a significant degree of sequence identity with the type I CaaX proteases such as Saccharomyces cerevisiae Ste24p and Caenorhabditis elegans CeFACE-1. A comparative phylogenetic analysis demonstrated that this protein family is tightly conserved across taxa, from bacteria to mammals. The bacterially expressed recombinant TsSte24p showed proteolytic activity, with an optimal pH of 7.5. The enzyme activity was significantly inhibited by EDTA. Its activity was increased in the presence of low concentrations of the Zn2+(0.001-0.01 mM); but was reversibly down-regulated at high doses (over 0.1 mM). The native TsSte24p appeared to function as a homodimer, the subunits of which were linked to each other via covalent disulfide bond. The protein was localized in the bladder wall and scolex with differential patterns of distribution. Our results indicated that TsSte24p is a zinc-dependent metalloprotease, which belongs to the FACE-1/Ste24p protease family.

[Tissue expansion under the cicatrix].

OBJECTIVE: To investigate a more simple and effective method to repair cicatrix by tissue expansion. METHODS: The dilator with the capacity of 80 - 500 ml was implanted into the subcutaneous pocket under the cicatrix. After dilating for one to two months, the dilator was taken out and the wound surface of the cicatrix was removed. The expanded skin flap was advanced or rotated to cover the defects. The procedure was used on 203 cases. RESULTS: The dilatation was achieved successfully in all the cases, followed by cicatrix removing and repair. The incision scar was not noticeable. CONCLUSIONS: Tissue expansion under the cicatrix has the advantages of safety, less trauma and less extra incisions. It is a reasonable choice to obtain more flexible surgical designs and more economical skin flap applications. It is suitable for most of the treatment for cicatrix.

[Full face dermabrasion for acne scars].

OBJECTIVE: Dermabrasion has been of great value in plastic surgery. Dermabrasion was developed for a specific indication; however, within a very short time, the concept of dermabrasion found wide applicability. This study was to investigate the application of dermabrasion in the treatment of acne scars. METHODS: From Feb. 1996 to May 2004, a total of 110 patients with acne scar were treated with dermabrasion. RESULTS: Postoperatively, the curative results were achieved in 45 cases; good results in 40 cases and effective results in 25 cases. The study revealed that the patients at 18-46 years of age have good results. CONCLUSIONS: Dermabrasion is a good and safe technique to treat the scar of acne.

[Minimizing the antigenicity of tracheal allografts by using a technique of cryopreservation].

OBJECTIVE: To investigate the proper time of cryo-preserving tracheal allograft so as to minimize its antigenicity. METHODS: On a dog model, this study was carried out by allografting a tracheal into a muscular flap formed with sternocephalic muscle and sternohyoid--sternothyroid muscle. The tracheal was treated with cryopreservation in defferent intervals. The viability of the graft was evaluated by the examination of fiberoptic bronchoscopy, histopathology and microangiography. The blood flow of the tracheal mucous was measured with a blood flowmeter and the survival area was decided in the calculation of the percentage. RESULTS: There are no significant differences in the mucous membrane appearance and the mucosal blood flow one week after the surgery among the non-cryopreservation group and the groups treated with cryopreservation in 1 day, 2 weeks, 4 weeks, 6 weeks and 8 weeks. The graft was found to start necrosis 2 weeks after the transplantation with the infiltration of mononuclear cells examined under light microscope in almost all of the groups, especially in the non-cryopreservation group and the groups treated with cryopreservation in 1 day, 2 weeks. However, there was no significant difference among the autograft group and the allograft groups cryopreservated in 6 weeks and 8 weeks, and the infiltration of the mononuclear cells was not found in these groups either. CONCLUSION: The antigenicity of the tracheal allografts could be significantly decreased by the treatment of cryopreservation over 6 weeks.

[Cleft lip nasal deformity corrected by an alar flap and the alar cartilage sling method].

OBJECTIVE: To search an effective method to correct the secondary nasal deformity. METHODS: The "spilth" tissue asymmetry to the another side on the cleft side alar is formed as a flap, which is used to drive up or reconstruct the nostril base (sill), readjust nostril size and shape. The cleft side alar cartilage lateral foot is disassociated, replaced and fixed into the normal place. RESULTS: Nineteen patients were received this operation, their nasal alar, nostril, sill, on the two sides are symmetry, and the result is good. CONCLUSIONS: The cleft side alar flap and alar cartilage sling procedure is effective to correct secondary cleft lip nasal deformity.

[The investigation of tracheas transplantation by wrapping in a muscle flap].

OBJECTIVE: To investigate the possibility of tracheas transplantation by wrapping it in a muscle flap. METHODS: With a dog model, a number of tracheas were separately wrapped in the unilateral sternocephalic muscle flap and the bilateral sternohyoid-sternothyroid muscle flap, and placed in the original site. The tracheas autografting was used as a control. The viability was evaluated by the examination of fiberoptic bronchoscopy, histopathology and microangiography, the measurement of tracheal mucosal blood flow and the calculation of survival rate and percentage of patency. RESULTS: The submucosal blood flow of the transplanted tracheas was detected in the unilateral sternocephalic muscle flap group and the bilateral sternohyoid-sternothyroid muscle flap group 1 week after the surgery and gradually reached the level close to the normal in 4 weeks, while the vascular ingrowth was also shown from the wrapped muscle flap into the transplanted tracheas by using a microangiography technique. The histopathological examination demonstrated that the structure of the transplanted tracheas was quite same as the original one and its inner surface was also covered with pseudostratified columnar ciliary epithelia. However, in the control group, the mucous membranes turned black one week after the transplantation and all dogs died from the graft necrosis. CONCLUSION: The tracheas wrapped in a muscular flap could survive well for a long time.