Insulin secretion dynamics of free and alginate-encapsulated insulinoma cells

This study investigates the effect of alginate/poly-l-lysine/alginate (APA) encapsulation on the insulin secretion dynamics exhibited by an encapsulated cell system. Experiments were performed with the aid of a home-built perfusion apparatus providing a 1 min temporal resolution. Insulin profiles were measured from: (i) murine insulinoma βTC3 cells encapsulated in calcium alginate/poly-l-lysine/alginate (APA) beads generated with high guluronic (G) or high mannuoric (M) content alginate, and (ii) murine insulinoma βTC-tet cells encapsulated in high M APA beads and propagated in the presence and absence of tetracycline. Results show that encapsulation in APA beads did not affect the insulin secretion profile shortly post-encapsulation. However, remodeling of the beads due to cell proliferation affected the insulin secretion profiles; and inhibiting remodeling by suppressing cell growth preserved the secretion profile. The implications of these findings regarding the in vivo function of encapsulated insulin secreting cells are discussed.     

鸡输卵管上皮细胞体外分离培养的研究

鸡输卵管上皮细胞是卵清蛋白的主要分泌细胞,是研究输卵管特异表达蛋白调控的重要工具。在以往的研究中,多采用普通DMEM培养液对鸡输卵管上皮细胞进行分离与培养,容易造成其自身特性在体外培养过程中的改变。本研究我们优化了细胞分离方法,发现从输卵管漏斗部组织分离的输卵管上皮细胞增殖较快;用鸡输卵管上皮细胞培养基相比DMEM更适合促进细胞生长;与胰酶相比,用Accutase消化酶进行细胞传代,有利于输卵管上皮细胞特性维持。对所获得的输卵管上皮细胞鉴定发现,己烯雌酚能促进卵清蛋白的表达,说明分离培养的细胞保持了鸡输卵管上皮细胞特性。本研究建立的方法为输卵管特异表达蛋白调控以及家禽生物反应器的研究奠定了基础。     

Nine surface plasmon resonance assays for specific protein quantitation during cell culture and process development

Quantitation of protein is essential during pharmaceutical development, and a variety of methods and technologies for determination of total and specific protein concentration are available. Here we describe the development of a streamlined assay platform for specific quantitation assays using surface plasmon resonance (SPR) technology. A total of nine different assays were developed using similar conditions, of which eight assays were for quantitation of different human blood plasma proteins (IgG, IgG1–4 subclasses, IgA, transferrin, and albumin) from a chromatography-based IgG plasma process. Lastly, an assay for monitoring the concentration of a recombinant monoclonal antibody during 13 days of CHO cell culturing was developed. Assay performances were compared with enzyme-linked immunosorbent assay (ELISA), nephelometry, ARCHITECT, and Cobas c501. SPR assays were shown to have higher sensitivity than analysis using nephelometry, ARCHITECT, and Cobas and to have significantly lower analysis and hands-on time compared with ELISA. Furthermore, the SPR assays were robust enough to be used for up to 12 days, allowing specific protein concentration measurement of a sample to be completed at line within 10 min. Using the same platform with only few varied parameters between different assays has saved time in the lab as well as for evaluation and presentation of results.     

Developmental changes in amniotic and allantoic fluid insulin-like growth factor (IGF)-I and -II concentrations of avian embryos

In the literature, IGFs in the developing embryo are usually determined by blood serum concentrations. For this study, IGF-I/-II was quantified in the amniotic and allantoic fluids of fertile commercial broiler chicken (Gallus domesticus) (n=222), Pekin duck (Anas platyrhyncha) (n=250), and turkey (Meleagridis gallopavo) eggs (n= 200) during incubation. Amniotic and allantoic fluids were collected from embryos starting at 6 days of incubation for chickens and 8 days of incubation for ducks and turkeys. IGF concentrations within the fluids were determined by radioimmunoassay. Chicken amniotic IGF-I concentration at stage 29 of development was significantly higher (P< or =0.05) than the duck or turkey. At stage 36 of development the concentration of IGF-II in the amniotic fluid was 2.8 times greater in the chicken versus the duck (P< or =0.05) and 2 times greater than in the turkey (P< or =0.05). Within species, chicken IGF-I concentration in the amniotic fluid had a cubic trend (P< or =0.001), duck IGF-I increased linearly (P< or =0.001), and turkey concentrations declined quadratically (P< or =0.001) throughout development. In all species, the IGF-II concentration was higher than the IGF-I concentration in the amniotic and allantoic fluids.     

与麦芽糖结合蛋白构象有关的单克隆抗体的制备

利用麦芽糖结合蛋白（ＭＢＰ）在大肠杆菌中的高效表达,分别采用亲和层析和变性复性,疑胶过滤的方法纯化了可溶性和包涵体部分的ＭＢＰ。将得到的这两种构象不同的ＭＢＰ分别免疫小鼠并进一步筛选和克隆,各得到５株单克隆抗体株。经制备腹水获得抗体后,地其和构象不同的麦芽糖结合蛋白的结合能力的测定,发现由包涵体部分的ＭＢＰ轩和筛选得到的单克隆抗体中有两对变性蛋白具有更高的结合能力。     

2002年北戴河林栖迁徙鸟类环志

2002年4月1日～10月31日,共环志林栖迁徙鸟类7目、20科、84种12244只,环志数量超过100只的鸟有23种。春季性别鉴定16种1224只,雌雄比例约为1：1．1;秋季鉴定20种1656只,雌雄比例约为1．4：1。春秋两季大致3d一个迁徙高峰,春季的迁徙曲线高峰斜度较秋季的大,秋季的迁徙高峰比较平缓。     

Cadmium-binding to transferrin in the plasma of the common carp Cyprinus carpio

In this study, it was investigated by autoradiography with radioactive cadmium after Western blotting of two-dimensional electrophoresis gels, to which proteins cadmium is mainly bound in plasma of common carp Cyprinus carpio. The obtained results demonstrate that in carp plasma, cadmium is primarily bound to two high molecular weight proteins. Relative small amounts are bound to a protein with M(r) approximately 60000. The other metal-binding protein, with M(r) approximately 70000 and pI approximately 6.7 was identified as transferrin. The conditional equilibrium constants for the binding of cadmium ions to the two metal-binding sites of this protein were calculated as logK(1)=5.40+/-0.12 and logK(2)=4.66+/-0.21, which are comparable to those of human transferrin under the same experimental conditions. Transport of cadmium in plasma of carp was found to be different from that of brown trout Salmo trutta and man, where cadmium is mainly bound to albumin and transferrin. The prominent binding of cadmium to transferrin can be explained by the absence or at least the very low concentrations in which albumin is present in carp plasma.     

Interaction of VO ion with human serum transferrin and albumin

The complexation of VO²⁺ ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO²⁺ ion occupies three different binding sites, A, B₁ and B₂, distinguishable in the X-band anisotropic spectrum recorded in D₂O. With albumin the results show that a dinuclear complex (VO)₂^dHSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO²⁺, the multinuclear complex (VO)_x^mHSA is the prevalent species, where x = 5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named “strong” site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the ⁵¹V A_z values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO²⁺/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO²⁺ concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.     

鸡鸭异种间嵌合体的制备

本研究采集孵化14期鸡胚血液中的原始生殖细胞,在液氮中冷冻保存三个月后,解冻成活率达80％以上。以Ficoll密度梯度离心将其从血液中分离纯化,分离获得纯度为27.5%,平均每枚胚胎可获得近50个PGCs。将分离的PGCs 100-200个以微注射法转移至15期早期麻鸭胚胎中制备了鸡鸭种间嵌合体,孵出8（6♂,2♀）只雏鸭,总孵化率为7.3%（8／110）。以鸡W染色体特异性DNA探针原位杂交法在早期鸭胚性腺中检测鸡PGCs。在被检测的21个鸭胚的性腺中,16个有不同程度的阳性信号,嵌合率达76.2%(16/21)。实验结果表明鸡原始生殖细胞能够迁移并定居到鸭胚性腺中,并有可能在鸭性腺中增殖分化成有功能的配子。     

Nonequivalence of second virial coefficients from sedimentation equilibrium and static light scattering studies of protein solutions

Experimental data for ovalbumin and lysozyme are presented to highlight the nonequivalence of second virial coefficients obtained for proteins by sedimentation equilibrium and light scattering. Theoretical considerations confirm that the quantity deduced from sedimentation equilibrium distributions is B(22), the osmotic second virial coefficient describing thermodynamic nonideality arising solely from protein self-interaction. On the other hand, the virial coefficient determined by light scattering is shown to reflect the combined contributions of protein-protein and protein-buffer interactions to thermodynamic nonideality of the protein solution. Misidentification of the light scattering parameter as B(22) accounts for published reports of negative osmotic second virial coefficients as indicators of conditions conducive to protein crystal growth. Finally, textbook assertions about the equivalence of second virial coefficients obtained by sedimentation equilibrium and light scattering reflect the restriction of consideration to single-solute systems. Although sedimentation equilibrium distributions for buffered protein solutions are, indeed, amenable to interpretation in such terms, the same situation does not apply to light scattering measurements because buffer constituents cannot be regarded as part of the solvent: instead they must be treated as non-scattering cosolutes.     

Interspecies variation in yolk selenium concentrations among eggs of free-living birds: The effect of phylogeny

Birds deposit the trace element selenium (Se) into their eggs because an adequate supply of this micronutrient is essential for embryonic development. Although there is considerable interest in egg Se with regard to topics as diverse as poultry nutrition and environmental pollution, data on the natural levels of Se in eggs of free-living avian species are currently very limited. To address this lack of information, we measured the yolk Se concentrations in eggs of 14 avian species collected in the wild. The concentrations (ng/g wet yolk) varied from 394 to 2238, with a mean value of 1040. Values (means+/-SD) for eggs from the UK, Canada and New Zealand were, respectively, 522+/-192 (3 species), 1194+/-584 (8 species) and 1147+/-200 (3 species). However, analysis by appropriate statistical models indicates that the effect of phylogenetic relatedness among these species is so significant that it removes any effect of geographical location. In particular, species belonging to the order Passeriformes displayed significantly higher yolk Se levels than Non-Passeriforme species. In marked contrast to the free-living species, our previously published data indicate that the Se concentration in egg yolk of the domestic chicken is only about 100 ng/g wet yolk when the birds are maintained on a basal commercial diet without supplementary Se. The results reveal an extensive interspecies variation in yolk Se (across a 6-fold range) for eggs collected from the wild. Nevertheless, the Se concentrations in the yolks of all the free-living species were far higher (4-21-fold) than that achieved in the yolk of the domestic chicken consuming a standard basal diet.     

四川南充高坪机场秋冬季节鸟类多样性与鸟撞预防

2006年9月-2007年2月,对四川南充高坪机场及其周围4种生境(草地、农耕区、村囤、松林)的鸟类进行调查,并进行鸟类多样性指数分析.结果共记录到鸟类81种,隶属于13目37科;秋冬季农耕区和村囤的鸟类密度、数量明显比草地和松林高;机场草地冬季鸟类的密度明显比秋季高.结合鸟类的生活习性及其栖息生境、Ⅳ值、分布广度和在各生境中的优势密度和总范围里的密度,秋季对鸟撞最具危险的是白鹭、家鸽、山斑鸠、白鹡鸰、白头鹎、小云雀、小鹀等;冬季对鸟撞最具威胁的是家鸽、小云雀、小鹀、金翅雀、白头鹎、水鹨等.并相应提出了相应一些的预防措施,如减少结实的植物、剪草降低草高、控制土壤动物的密度等.     

NORTH AMERICAN GRASSLAND BIRDS: AN UNFOLDING CONSERVATION CRISIS?

Abstract: The widespread and ongoing declines of North American bird populations that have affinities for grassland and grass-shrub habitats (hereafter referred to as grassland birds) are on track to become a prominent wildlife conservation crisis of the 21st century. There is no single cause responsible for the declines of grassland birds. Rather, a cumulative set of factors such as afforestation in the eastern United States, fragmentation and replacement of prairie vegetation with a modern agricultural landscape, and large-scale deterioration of western U.S. rangelands are the major causes for these declines. The North American Bird Conservation Initiative (NABCI) is a set of comprehensive and coordinated strategic actions modeled on the Joint Venture initiatives that were used to successfully implement the North American Waterfowl Management Plan. The NABCI is emerging as a potential broad-scale solution for conserving populations of grassland birds. Coordinating grassland bird conservation efforts with initiatives to stabilize and increase upland game birds that have strong affinities for grassland habitats—such as quail and prairie grouse—presents additional opportunities to leverage funding and resources that will positively impact virtually all species of North American grassland birds.     

The sequence-immunology correlation revisited: Data for cetacean myoglobins and mammalian lysozymes

Quantitative microcomplement fixation tests employing rabbit antisera were done to compare immunologically 13 cetacean myoglobins and 15 mammalian lysozymes c of known amino acid sequence. In both cases there was a strong correlation between immunological distance (y) and percent sequence difference (x), as had been found for several other globular proteins. For myoglobin the relationship could be described by y = 10.5x and for lysozyme by y = 8.5x. The coefficients in both of these equations are appreciably higher than the values of 5.1–6.9 reported for three other vertebrate globular proteins (bird lysozyme c, mammalian ribonuclease, and mammalian serum albumin), and they imply that rabbit antisera to mammalian myoglobins and lysozymes are more sensitive to evolutionary substitutions. A strong inverse correlation (r = -0.95) was found when the slope of the line relating y to x for these five data sets was plotted against the percent sequence difference between the rabbit's own protein and the proteins immunized with. Specifically, the cetacean myoglobins on average differ in amino acid sequence from rabbit myoglobin by less than 13% and exhibit the steepest slope (10.5), while bird lysozyme sequences differ by nearly 40% from rabbit lysozyme and exhibit the shallowest slope (5.1).     

The evolution of avian mating systems: a phylogenetic analysis of male and female polygamy and length of pair bond

A phylogenetic analysis of the evolution of mating systems in birds was made, based on the phylogeny of Sibley and Ahlquist. Both the mating behaviour and the social behaviour of species were classified for males and females, according to (1) the frequency of mated individuals in a species having several mates in a breeding season compared to those having one mate (the mating pattern), and (2) whether there is a bond between males and females, and in case of there being a bond, to the length of the bond. Non-passerines are extensively analysed, whereas we only give a survey of the situation in passerines. In non-passerine birds, the number of inferred transitions from monogamy to polygamy are 15 for females and 16–23 for males. Almost all transitions between different states of mating pattern are to higher states of polygamy. Our analyses also show a concentration of transitions to polygamy and short bonds, respectively, in the two monophyletic groups of Struthionidae-Anatidae and Pteroclidae-Laridae.