Improved suppression of circulating complement does not block acute vascular rejection of pig-to-rhesus monkey cardiac transplants

At present, acute vascular rejection (AVR) remains a primary obstacle inhibiting long-term graft survival in the pig-to-non-human primate transplant model. The present study was undertaken to determine whether repetitive injection of low dose Yunnan-cobra venom factor (Y-CVF), a potent complement inhibitor derived from the venom of Naja kaouthia can completely abrogate hemolytic complement activity and subsequently improve the results in a pig-to-rhesus monkey heterotopic heart transplant model. Nine adult rhesus monkeys received a heterotopic heart transplant from wild-type pigs and the recipients were allocated into two groups: group 1 (n = 4) received repetitive injection of low dose Y-CVF until the end of the study and group 2 (n = 5) did not receive Y-CVF. All recipients were treated with cyclosporine A (CsA), cyclophosphamide (CyP) and steroids. Repetitive Y-CVF treatment led to very dramatic fall in CH50 and serum C3 levels (CH50 < 3 units/C3 remained undetectable throughout the experiment) and successfully prevented hyperacute rejection (HAR), while three of five animals in group 2 underwent HAR. However, the continuous suppression of circulating complement did not prevent AVR and the grafts in group 1 survived from 8 to 13 days. Despite undetectable C3 in circulating blood, C3 deposition was present in these grafts. The venular thrombosis was the predominant histopathologic feature of AVR. We conclude that repetitive injection of low dose Y-CVF can be used to continuously suppress circulating complement in a very potent manner and successfully prevent HAR. However, this therapy did not inhibit complement deposition in the graft and failed to prevent AVR. These data suggest that using alternative pig donors [i.e. human decay accelerating factor (hDAF)-transgenic] in combination with the systemic use of complement inhibitors may be necessary to further control complement activation and improve survival in pig-to-non-human primate xenotransplant model.

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 mug/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 mug/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB2 and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 mug/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB2 in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.

Fridericia liangi sp nov (Enchytraeidae, Oligochaeta) from Changbaishan Mountain, China

We describe one new enchytraeid species, Fridericia liangi sp. nov., from Mt. Changbaishan, Jilin Province, northeastern China. It was collected from soils at the foot of Changbaishan Mountain and is distinguished from all known congeners by the following combination of characters: 1) no lateral chaetae, only ventral chaetae throughout, 2) a maximum of four chaetae in ventral preclitellar bundles, 3) one chaeta in ventral postclitellar bundles, 4) dorsal pores from VII on, 5) esophageal appendages unbranched, 6) coelomocytes without refractile vesicles, 7) clitellum girdle shaped, well developed, 8) no subneural glands, and 9) spermathecae simple.

Three new species of Tubificinae, Oligochaeta, from two plateau lakes in Southwest China

Three new species of Tubificinae (Naididae, Oligochaeta), Varichaetadrilus vestibulatus n. sp., Aulodrilus apeniatus n. sp., and Ilyodrilus mesoprostatus n. sp., are reported from Fuxian Lake and Xingyun Lake of Yunnan Province, Southwest China. V. vestibulatus differs from its allies by possessing modified spermathecal chaetae and thinner cylindrical penial sheaths. A. apeniatus is unique in the genus by having no penis. I. mesoprostatus is distinguishable from congeners by its prostate glands joining middle portion of atria and having concave, cone-shaped cuticular penial sheaths. Twenty-eight species of freshwater oligochaetes have hitherto been recorded from Yunnan Province, including five endemic species from three plateau lakes.

Fridericia dianchiensis sp nov (Enchytraeidae, oligochaeta) from Yunnan Province, China

Fridericia dianchiensis, a new enchytraeid species collected from Yunnan Province, is described here. It is characterized by a combination of the following characters: 1) lateral bundles containing maximum 3 chaetae; 2) esophageal appendages with 3-4 simple, terminal branches; 3) dorsal vessel originating in XX-XXIII; 4) sub-neural glands absent; 5) seminal vesicle large, occupying two segments; 6)clitellum girdle-shaped or gland cells absent between bursal slits and pre-middle ventrally; 7) coelomocytes without refractile vesicles, 8) spermatheca without diverticula and both ampullae broadly united; and 9) long spermathecal ectal duct without gland at the orifice.

Redescription of Nais inflata Liang (Annelida : Oligochaeta) from China

Nais inflata Liang 1963 is redescribed on the basis of fully mature specimens collected from several localities of the Yangtze River and Yellow River. The observations and redescription resulting from the study of mature specimens supports N. inflata as a valid species. This species is characterized by a thickened body, the ventral crotchets all of the same type with distal tooth usually longer than proximal one, a greater number of dorsal chaetae per bundle, stout dorsal needles with equal minute bifid teeth (<1 mu m), presence of penial chaetae, and prostate glands on vasa deferentia instead of atria. The species is most closely related to N. communis Piguet, 1906 and N. variabilis Piguet, 1906. A table comparing allied species is provided.

Potamothrix scleropenis sp nov (Oligochaeta : Tubificidae) from Fuxian Lake, the deepest lake in southwest China

Potamothrix scleropenis sp. nov. (Tubificidae: Tubificinae) is described from the profundal zone (74 m) of Fuxian Lake, the deepest lake (up to 155 m) on the Yunnan-Guizhou Plateau in China. The new species is assigned to Potamothrix because of its short vasa deferentia. and its tubular atria without ejaculatory ducts and prostate glands. It differs from congeners by its cuticularized penis sheaths; bifurcated, strongly curved spermathecal chaetae; bifurcated lower prongs of bifids; and feathered hairs. P scleropenis appears closely related to P cekanovskajae Finogenova, 1972 and P tudoranceai porka, 1994, since all the three species have homogeneous atria without prostate glands.

Bryodrilus fuscistriatus, a new enchytraeid species (Oligochaeta : Enchytraeidae) from northeastern China

Bryodrilus fuscistriatus, a new enchytraeid species from Mt. Changbaishan, Jilin Province, north-eastern China, is described. It is characterized by brown epidermal glands, 7 pairs of preclitellar nephridia, poorly-developed clitellar glands, spermatheca with 2 sessile globular diverticula, and a long sperm funnel with a very broad collar. It is similar to the Alaskan B. tunicatus Dozsa-Farkas & Christensen, 2002 in possessing two diverticula in the spermathecal ampulla and the origin of the dorsal vessel, and the Chinese B. longifistulatus and B. macrotheca Xie et al., 2000c in body size, long sperm funnel and undeveloped clitellar glands, but it differs from B. tunicatus by the presence of brown-striped epidermal gland cells in III-V, a poorly-elevated clitellum, the absence of copulatory glands in XIII-XIV, the regular outline of coelomocytes, and 7 pairs of preclitellar nephridia; from B. longifistulatus and B. macrotheca by the shape of spermatheca, the color of epidermal gland cells, the position of the first pair nephridia, and the origin of dorsal vessel.

The precocious Chinese mitten crab: Changes of gonad, survival rate, and life span in a freshwater lake

Changes of the gonad. survival rate, and life span of the precocious (i.e., sexually mature in their first October-November, at the age of 5 or 6 months) Chinese mitten crab (Eriocheir sinensis) were studied in a small, shallow, macrophytic, freshwater lake along the middle reaches of the Changjiang River, China. The gonosomatic index (GSI) reached peak during the next March when the female GSI was 12.32 +/- 1.75 SD% and the male GSI A as 4.24 +/- 0.19%. The sexual glands degraded from then on. In the first ten-day period of the following July, there was no complete ovum in the ovary. and the sperm became thin and lost adhesion. The population declined sharply from November to July, and the last one (a female) died in the middle of July, which indicated that the life span of the precocious crab was about 12 months (from larval hatching in June to death in July of next year). The survival curve might be expressed as Y = 1.09exp(-0.018x) (Y survival rate; x: days) for the precocious crabs stocked in experimental cages.

Marine Phallodrilinae (Oligochaeta, Tubificidae)of Hainan Island in southern China

Six species of Phallodrilinae are reported from Hainan Island in southern China. The mesopsammic Aktedrilus yiboi sp. nov. is new to science. It is characterized by a combination of (1) small, straight, conical, strongly cuticularized penis sheaths, (2) posterior prostate glands being associated with the most ectal parts of the atria, and (3) small spermathecal ampulla. Bathydrilus ampliductus Erseus, 1997 is recorded for the first time since it was originally described from Australia's Northern Territory.

Four new terrestrial species of Marionina (Clitellata, Enchytraeidae) from China and re-examination of M. hoVbaueri Moller

Enchytraeid surveys were made in China, mainly along the Changjiang (Yangtze) River Basin, during the period 1991-1999. Among the findings, four terrestrial species of Marionina are new to science and well illustrate the taxonomic complexity of the genus as currently defined. Marionina sinica sp. n. is characterized by a specific chaetal distribution, the marionine pattern of the dorsal blood vessel, and elongate, fusiform, spermathecal ectal ducts. Marionina sacculata sp. n. is distinguished by the possession of a pair of pouch-like oesophageal appendages in IV, the lack of lateral chaetae in VII-XI, a marionine pattern of the dorsal blood vessel, and short spermathecal ectal ducts gradually expanding into spherical ampullae. Both M. sinica and M. sacculata have minute bodies (2-3 mm long in vivo) and lack spermathecal accessory glands. The former species shows its closest aYnities with the European M. brendae Rota, 1995, whereas the latter is closest to the German M. hoVbaueri Moller, 1971, for which an amended diagnosis is provided. Marionina seminuda sp. n. has only ventral chaetal bundles, distributed from III onwards and bisetose. It is similar to the Holarctic M. subterranea (Knollner, 1935) in lacking entirely the lateral chaetae and in having the brain incised posteriorly, the dorsal vessel bifurcating behind the pharynx, and coelomocytes containing opaque granules, but diVers from it in having the longest chaetae in preclitellar segments and gland cells distributed all over the spermathecal ectal ducts. Marionina righiana sp. n. is diagnosed by the location of the head pore on the prostomium, the absence of lateral chaetae from VIII ( VII or IX) onwards, the possession of free spermathecae extending backwards through one to four segments, the brain deeply incised posteriorly, the lumbricilline pattern of the dorsal blood vessel, and the opacity of coelomocytes in vivo. Prior to this study, members of the genus so atypical as M. righiana with respect to the position of the head pore and the structure of the spermathecae were known only from South American soils. Until the taxonomy of Marionina has been more thoroughly assessed and revised, the assignment of the four species to this large assemblage should be regarded as tentative.

Fridericia nanningensis, a new terrestrial enchytraeid species (Oligochaeta) from southwestern China

Fridericia nanningensis, a new species from wetland soil of Nanhu Park, Nanning city, the capital of Guangxi Zhuang Autonomous Region in southwest China, is described. It is characterized by 2-4 chaetae per bundle, poorly-developed clitellar glands, slender, unbranched peptonephridia, and spermathecae with 2 ampullar diverticula, a deep constriction in the middle of the ampulla and one large ectal gland. It is closely related to the European species, F. alata Nielsen & Christensen, 1959 and the East European species, F. tubulosa Dozsa-Farkas, 1972 by the shape of peptonephridia and the undeveloped clitellar glands. It differs from F. alata by its shorter body length and fewer chaetae per bundle, its type of coelomocytes (type "c"), its deep constriction in the middle of the spermathecae ampulla and a larger ectal gland, and it differs from F. tubulosa by its pale epidermal glands, its more anterior origin of the dorsal vessel, a deep constriction in the middle of the spermathecae ampulla, shorter ectal duct, and only one ectal gland at the spermathecal orifice.

蛇毒血管内皮生长因子及丝氨酸蛋白酶类似物的研究

蝰科蛇毒中含有丰富的具有血管通透增强活性的蛋白组分，本论文结合生物化学与分子生物学手段对几种毒蛇的蛇毒血管内皮生长因子进行了研究。其中，从云南产菜花烙铁头（Trimeresurus jerdonii）蛇毒中分离得到一个血管内皮生长因子，TjsvVEGF，并研究了其活性与受体结合特性的关系。同时，对圆斑蝰蛇、蝮蛇、山烙铁头和竹叶青等几种蛇的svVEGFs进行了分子生物学研究。此外，我们还分离到了一个蛇毒丝氨酸蛋白酶类似物,TjsvSPH,还对该蛋白的理化性质进行了初步研究。 TjsvVEGF是一个表观分子量为29 kDa的二聚体蛋白，由两个相同大小的亚基组成。活性实验表明，它在10ng的剂量下即可明显提高血管通透性，活性强度与VEGF165相当。虽然TjsvVEGF的氨基酸序列与TfsvVEGF和Pm-VEGF具有较高的相似性，但是它们的受体结合特性却有很大差异。TjsvVEGF与VEGFR-1的结合能力很弱，而对VEGR-2有很高的亲和力。这说明，TjsvVEGF的活性主要是VEGFR-2介导的。最后，我们对导致TjsvVEGF对VEGFR-1低亲和力的原因进行了探讨。 利用PCR方法，我们从圆斑蝰蛇毒腺中得到了三种svVEGFs蛋白编码区长短不一的cDNA序列，其差异是由cDNA链中一段富含AG（3’拼接接受位点）的区段发生了核苷酸缺失产生的。蛋白编码区核苷酸的缺失导致其编码的三种svVEGF蛋白N末端的氨基酸序列和长短均产生较大差异。因此我们推测，蝰属svVEGFs蛋白N末端普遍较短可能是编码它们的mRNA前体对3’拼接点的不同选择产生的。同时，通过对cDNAs推导的氨基酸序列分析发现，蝮蛇svVEGF和山烙铁头svVEGF在与受体结合相关的多个重要位点上发生了氨基酸替换，提示它们是研究svVEGFs与VEGFR结合机制的良好材料。 通过分子筛、离子交换和亲和层析等方法，我们还从菜花烙铁头蛇毒中得到了一个不具有酶活性的丝氨酸蛋白酶类似物，命名为：TjsvSPH。内肽序列测定结果表明，TjsvSPH三联体结构中的组氨酸已突变为精氨酸，这很可能是导致其失去蛋白水解酶活性的主要原因。活性检测验表明，与许多已发现的蛇毒活性组分不同，TjsvSPH不具有蛋白水解酶活性，不能引起血小板聚集，也不抑制ADP和凝血酶诱导的血小板聚集。它对人血浆的复钙时间也没有影响。TjsvSPH的氨基酸序列与典型蛇毒丝氨酸蛋白酶Halystase和Calobin有约77％的一致性。它与同科属来源的蛇毒丝氨酸蛋白酶类似物氨基酸序列相似性高达92％以上，与不同科属的也有约74％以上的相似性。通过对Genbank中六种毒蛇所有丝氨酸蛋白酶及其类似物进化分析，我们推测，蛇毒丝氨酸蛋白酶类似物很可能是由蛇毒丝氨酸蛋白酶进化而来，并在进化过程中形成了一类独特的蛋白质。

竹叶青蛇毒金属蛋白酶结构与功能的研究

出血性的蝰科蛇毒中含有丰富的蛇毒金属蛋白酶，本论文就竹叶青（Trimeresurus stejnegeri）蛇毒中的蛇毒金属蛋白酶的结构与功能进行研究。我们用生物化学手段从竹叶青（T.stejnegeri）的粗毒中分离纯化得到一个二聚体的P-IIIb亚型蛇毒金属蛋白酶，命名为TSV-DM。同时，用分子生物学方法从竹叶青（T.stejnegeri）的毒腺cDNA文库中克隆得到3个P-III型的蛇毒金属蛋白酶的cDNAs序列， 其中一个编码TSV-DM蛋白前体，另两个编码P-IIIc亚型的出血性蛇毒金属蛋白酶前体，分别命名为stejnihagin-A 和 stejnihagin-B。 经过阴离子层析和肝素亲和层析两步层析方法，我们从竹叶青（T. stejnegeri）蛇毒中分离纯化得到TSV-DM蛋白质，非还原条件下SDS-PAGE电泳表观分子量约110 kDa，还原条件下约为55 kDa。活性检测表明，TSV-DM降解牛纤维蛋白原Aα链快于Bβ链，且呈剂量依赖关系。但不降解明胶，不诱导出血，不具有促凝或者抗凝活性，以及不诱导或者抑制血小板聚集。蛋白质N-末端测序表明TSV-DM的成熟蛋白N-末端封闭。利用肽指纹图谱确证了TSV-DM的编码cDNA。TSV-DM的cDNA序列编码622个氨基酸残基的蛋白前体，包括信号肽、前肽、金属蛋白酶区域、间隔区、去整合素样区域和富含半胱氨酸区域。TSV-DM与其他P-III型蛇毒金属蛋白酶的一级结构序列比对发现TSV-DM和诱导血管内皮细胞凋亡的P-IIIb亚型蛇毒金属蛋白酶具有高度的同源性。但是用人脐带静脉血管内皮细胞系ECV304细胞作为靶细胞检测TSV-DM的诱导血管内皮细胞凋亡活性发现，TSV-DM只能抑制ECV304细胞的增殖和诱导细胞形态从多角形的内皮细胞向成纤维细胞样的梭形状改变。电泳检测抽提的片断化DNA以及流式细胞仪检测TSV-DM处理的ECV304细胞的DNA含量变化均表明TSV-DM不能诱导ECV304细胞的凋亡。 Stejnihagin-A 和stejnihagin-B是用PCR方法从竹叶青（T.stejnegeri）毒腺cDNA文库中克隆得到的两个P-III型蛇毒金属蛋白酶前体的cDNAs。这两个cDNA序列均编码600个氨基酸残基的蛋白前体，包括信号肽、前肽、金属蛋白酶区域、间隔区、去整合素样区域和富含半胱氨酸区域。推导成熟肽的氨基酸序列分析结果表明，stejnihagin-A 和stejnihagin-B不仅在一级结构序列上和来源于黄绿烙铁头（Trimeresurus flavoviridis）的HR1b具有高度同源性，高达79%， 而且在他们的金属蛋白酶区域的第100个氨基酸残基位置上均有一保守的半胱氨酸残基。结合序列比对和进化树的分析，我们推测stejnihagin-A、stejnihagin-B和HR1b有可能组成一个新的P-III型蛇毒金属蛋白酶亚型，命名为P-IIIc亚型。