Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial cells (HCE). Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell- attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favours the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

Genetic analysis of Indian tasar silkmoth (Antheraea mylitta) populations

Indian tasar silkmoth, Antheraea mylitta is an economically important wild silkmoth species distributed across India. A number of morphologically and ethologically well-defined ecotypes are known for this species that differ in their primary food plant specificity. Most of these ecotypes do not interbreed in nature, but are able to produce offspring under captive conditions. Microsatellite markers were developed for A. mylitta, and out of these, ten well-behaved microsatellite loci were used to analyze the population structure of different ecoraces. A total of 154 individual moths belonging to eight different ecoraces, were screened at each locus. Hierarchical analysis of population structure using Analysis of MOlecular VAriance (AMOVA) revealed significant structuring (F-ST = 0.154) and considerable inbreeding (F-IS = 0.505). A significant isolation by distance was also observed. The number of possible population clusters was investigated using distance method, Bayesian algorithm and self organization maps (SOM). The first two methods revealed two distinct clusters, whereas the SOM showed the different ecoraces not to be clearly differentiated. These results suggest that although there is a large degree of phenotypic variation among the different ecoraces of A. mylitta, genetically they are not very different, and the phenotypic differences may largely be a result of their respective ecology.

熊猫（Macaca assamensis）精子的电镜观察

用光镜、扫描电镜及透射电镜首次研究了熊猴精子的结构。在光镜及电镜下，熊猴精子头的形状、顶体大小、核的结构等，与猕猴属其他种类相似。精子总长（７９．４９±０．１１μｍ）及各部分的长度与藏酋猴相近，而与猕猴属其他种类存在明显差异（Ｐ＜０．０１）。本文的结果表明，熊猴可能在种系进化过程中与藏酋猴关系最近，而与猕猴属中其他种分离较早。

Engineered silk fibroin protein 3D matrices for in vitro tumor model

3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.

Incorporation of exogenous RGD peptide and inter-species blending as strategies for enhancing human corneal limbal epithelial cell growth on Bombyx mori silk fibroin membranes

While fibroin isolated from the cocoons of domesticated silkworm Bombyx mori supports growth of human corneal limbal epithelial (HLE) cells, the mechanism of cell attachment remains unclear. In the present study we sought to enhance the attachment of HLE cells to membranes of Bombyx mori silk fibroin (BMSF) through surface functionalization with an arginine-glycine-aspartic acid (RGD)-containing peptide. Moreover, we have examined the response of HLE cells to BMSF when blended with the fibroin produced by a wild silkworm, Antheraea pernyi, which is known to contain RGD sequences within its primary structure. A procedure to isolate A. pernyi silk fibroin (APSF) from the cocoons was established, and blends of the two fibroins were prepared at five different BMSF/APSF ratios. In another experiment, BMSF surface was modified by binding chemically the GRGDSPC peptide using a water-soluble carbodiimide. Primary HLE were grown in the absence of serum on membranes made of BMSF, APSF, and their blends, as well as on RGD-modified BMSF. There was no statistically significant enhancing effect on the cell attachment due to the RGD presence. This suggests that the adhesion through RGD ligands may have a complex mechanism, and the investigated strategies are of limited value unless the factors contributing to this mechanism become better known.

食蟹猴和熊猴杂种F_1的细胞遗传学研究

本工作采用染色体显带、睾丸组织学切片以及低渗铺张──硝酸银染色等方法较为详细地研究了食蟹猴和（Ｍ．ｆａｓｃｉｃｕｌａｒｉｓ）和熊猴（Ｍ．ａｓｓａｍｅｎｓｉｓ）种间杂种Ｆ_１体细胞染色体的Ｇ带、Ｃ带，Ａｇ－ＮＯＲ、精母细胞联会复合体的结构、形态和行为以及精子发生。结果表明：（１）杂种亲本的染色体组具有高度的同源性；（２）杂种的精子发生过程完全正常；（３）食蟹猴和熊猴的种间生殖屏障可能主要是生态隔离。此外，本文还对食蟹猴和熊猴分类地位以及杂种细胞中Ａｇ－ＮＯＲ的异态性等问题进行了分析和讨论。

猕猴属6个种的rDNA变异及其系统进化关系

以人28S,18S DNA为探针,用15种限制性内切酶构建了猕猴属6个种(M.mulatta、M.facsicularis、M.arctoides、M.assamensis、M.thibetana、M.nemestrina)和滇金丝猴(Rhinopithecus bieti),白颊长臂猿(Hylobates leucogenys)核糖体DNA重复单位的限制性内切酶图谱。红面猴(M.arctoides)与熊猴(M.assamensis)拥有完全相同的限制性内切酶图谱。基于内切酶图谱得到了68个信息位点并计算了各种rDNA重复型间的遗传距离。用PHYLIP version 3.5c软件包中的NEIGHBOR和RESTML程序,以滇金丝猴和白颊长臂猿为外群,构建了NJ树和最大似然树。两棵树的拓扑结构不完全一致,但恒河猴( (M.mulatta)和食蟹猴(M.facsicularis)总是位于树的基部。熊猴-红面猴(M.assamensis-M.arctoides)虽然与藏猴(M.thibetana)共享的限制性位点数更多,在NJ树上两类动物也最为接近,但在最大似然树中熊猴-红面猴却与平顶猴(M.nemestrina)聚在一起。因此,rDNA变异的数据尚不能对猕猴类动物进行有效的分组。

PHYLOGENETIC-RELATIONSHIPS OF MACAQUES AS INFERRED FROM RESTRICTION-ENDONUCLEASE ANALYSIS OF MITOCHONDRIAL-DNA

Mitochondrial DNAs (mtDNAs) purified from 25 samples of 6 species of macaques, Macaca mulatta, M. fascicularis, M. arctoides, M. nemestrina, M. assamensis and M. thibetana, were analyzed to study the phyletic relationships among the species. A total of 36-46 sites was observed in each sample. By combining the cleavage patterns for each of the endonucleases, the 25 samples were classified into 11 restriction types. When data on M. fuscata and M. cyclopis collected by other authors were added to our own, the resultant molecular phylogenetic trees indicated that the 8 species may be divided into 4 groups: (1) M. mulatta, M. fuscata, M. cyclopis and M. fascicularis; (2) M. arctoides, (3) M. nemestrina; (4) M. assamensis and M. thibetana. Our results suggest that within both the fascicularis and sinica groups genetic distances are small between members and that the status of the species within the groups may require further investigation.

Sequence evolution of the CCR5 chemokine receptor gene in primates

The chemokine receptor CCR5 can serve as a coreceptor for M-tropic HIV-1 infection and both M-tropic and T-tropic SIV infection. We sequenced the entire CCR5 gene from 10 nonhuman primates: Pongo pygmaeus, Hylobates leucogenys, Trachypithecus francoisi, Trachypithecus phayrei, Pygathrix nemaeus, Rhinopithecus roxellanae, Rhinopithecus bieti, Rhinopithecus avunculus, Macaca assamensis, and Macaca arctoides. When compared with CCR5 sequences from humans and other primates, our results demonstrate that:(1) nucleotide and amino acid sequences of CCR5 among primates are highly homologous, with variations slightly concentrated on the amino and carboxyl termini; and (2) site Asp13, which is critical for CD4-independent binding of SIV gp120 to Macaca mulatta CCR5, was also present in all other nonhuman primates tested here, suggesting that those nonhuman primate CCR5s might also bind SIV gp120 without the presence of CD4. The topologies of CCR5 gene trees constructed here conflict with the putative opinion that the snub-nosed langurs compose a monophyletic group, suggesting that the CCR5 gene may not be a good genetic marker for low-level phylogenetic analysis. The evolutionary rate of CCR5 was calculated, and our results suggest a slowdown in primates after they diverged from rodents. The synonymous mutation rate of CCR5 in primates is constant, about 1.1 x 10(-9) synonymous mutations per site per year. Comparisons of K-a and K-s suggest that the CCR5 genes have undergone negative or purifying selection. K-a/K-s ratios from cercopithecines and colobines are significantly different, implying that selective pressures have played different roles in the two lineages.

Phylogenetic relationships of the macaques (Cercopithecidae : Macaca), inferred from mitochondrial DNA sequences

To study the phylogenetic relationships of the macaques, five gene fragments were sequenced from 40 individuals of eight species: Macaca mulatta, M. cyclopis, M. fascicularis, M. arctoides, M. assamensis, M. thibetana, M. silenus, and M. leonina. In addition, sequences of M. sylvanus were obtained from Genbank. A baboon was used as the outgroup. The phylogenetic trees were constructed using maximum-parsimony and Bayesian methods. Because five gene fragments were from the mitochondrial genome and were inherited as a single entity without recombination, we combined the five genes into a single analysis. The parsimony bootstrap proportions we obtained were higher than those from earlier studies based on the combined mtDNA dataset. Excluding M. arctoides, our results are generally consistent with the classification of Delson (1980). Our phylogenetic analyses agree with earlier studies suggesting that the mitochondrial lineages of M. arctoides share a close evolutionary relationship with the mitochondrial lineages of the fascicularis group of macaques (and M. fascicularis, specifically). M. mulatta (with respect to M. cyclopis), M. assamensis assamensis (with respect to M. thibetana), and M. leonina (with respect to M. silenus) are paraphyletic based on our analysis of mitochondrial genes.

SEROLOGICAL SURVEY OF A CAPTIVE MACAQUE COLONY IN CHINA FOR ANTIBODIES TO SIMIAN TYPE-D RETROVIRUSES

Sera from 510 macaques consisting of Macaca mulatta, Macaca assamensis, Macaca fascicularis, Macaca nemestrina, and Macaca arctoides were investigated for antibodies to simian AIDS type D retrovirus (SRV) by ELISA and Western blot with viral antigens purified from supernatants of SRV-1 infected cell cultures. Of these monkeys, 104 were seropositive by ELISA; only 23 were confirmed by Western blot. The true positive reaction to SRV was found in 15 of 463 (3.2%) M. mulatta and eight of eleven (72.7%) M. assamensis.

中国猕猴类(Macaca)的演化

对6种中国分布猕猴某些头骨特征的比较和35个比例性状的聚类分析, 并对 这几个种的亲缘关系进行了探讨。结果表明: M. assamensis和M. thibetana.M. mulatta和M. cyclopis关 系最为接近, 在前两者相聚以后, 与M. nemestrina相 聚成一大支系, 后两者与M. arctoides相聚成另一大支系, 最后两大支系聚在 一起成为中国猕猴类的亲缘关系。图1表1参21

中国熊猴的分类整理

该文通过查看国内收藏的标本对 M.assamensis 进行了分类整理, 认为 M.a.coolidgei 的亚种地位应予恢复, 滇西地区熊猴可能代表一个新类群. 通过t检验, 滇南和越南等地与滇西北和西藏东南部等地熊猴头骨的某些特征表现出显著或极显著差异. 作为区分 coolidgei 与 assamensis 的指标: coolidgei 体型较小, 肩背毛较短, 35—75mm, 背毛环纹略显或不明显, 0—2环, 体色更为灰暗; assamensis 体型较大, 肩背毛较长, 85—110mm, 背毛环纹明显, 3—4环。

乳酸脱氢酶 LDH 同功酶在猕猴属四种动物正常血清中的含量比较

用聚丙烯酰胺凝胶电泳 PAGE 和瑞典LKB-2202型激光光密度扫描仪, 分析测定了猕猴属中恒河猴(Macacamulatta), 红面猴(M.arctiodes), 熊猴(M.assamensis)和平顶猴(M.nemestrina)等四个种正常血清乳酸脱氢酶(LDH)同功酶谱型及含量变化. 结果表明: 这四个种正常血清的LDH同功酶及A、B两种亚基的含量存在着差异. 其中恒河猴与平顶猴之间的差异相对较大, 熊猴与红面猴之间的差异相对较小. 该文讨论了这些差异与这四种猴的分类地位的关系。