Isolation and characterization of gap junctions from tissue culture cells.

The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 A resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least 22 A. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

ConA的抗着床效应

本文用凝集素为探针,探索糖复合物在胚泡着床中的作用,报道了与甘露糖苷有专一结合的伴刀豆凝集素(ConA)有明显的抗小鼠胚泡着床作用。妊娠4d的小鼠,每只子宫角中注入Con A 25μg,22只子宫角中只有4只子宫角有胚泡着床,着床率为18.2％,与生理盐水对照组的着床率87.5％相比有明显差异。将相同剂量的Con A先与0.4mol/L α-甲基-D-甘露糖苷温育1—2h后再注入子宫,20只子宫角中有15只子宫角有胚泡着床,着床率提高到75％。用辣根过氧化物酶直接标记法证明,着床前子宫内膜细胞表面有Con A受体存在,并随着妊娠天数而增加,尤其是间质细胞,发情期时时为阴性反应,到着床期蜕膜细胞膜表面呈现出大量Con A受体。提示精复合物在着床中的重要作用。与甘露糖苷同样专一结合的,但寡糖结构专一性与Con A不同的豌豆凝集素注入子宫则无抗着床效应,着床率为85.7％。由此可以推测,N-连接的包含二个未被取代的或只在C-2位被取代的α-甘露糖苷的寡糖参于胚泡与子宫内膜相互作用的着床过程。     

Description and quantification of pteropod shell dissolution: a sensitive bioindicator of ocean acidification

Anthropogenic ocean acidification is likely to have negative effects on marine calcifying organisms, such as shelled pteropods, by promoting dissolution of aragonite shells. Study of shell dissolution requires an accurate and sensitive method for assessing shell damage. Shell dissolution was induced through incubations in CO₂‐enriched seawater for 4 and 14 days. We describe a procedure that allows the level of dissolution to be assessed and classified into three main types: Type I with partial dissolution of the prismatic layer; Type II with exposure of underlying crossed‐lamellar layer, and Type III, where crossed‐lamellar layer shows signs of dissolution. Levels of dissolution showed a good correspondence to the incubation conditions, with the most severe damage found in specimens held for 14 days in undersaturated condition (Ω ~ 0.8). This methodology enables the response of small pelagic calcifiers to acidified conditions to be detected at an early stage, thus making pteropods a valuable bioindicator of future ocean acidification.     

Interaction of light and temperature on seed germination of Rumex obtusifolius L.

Germination of Rumex obtusifolius L. seeds (nutlets) is low in darkness at 25° C. Germination is stimulated by exposure to 10 min red light (R) and also by a 10-min elevation of temperature to 35° C. A 10-min exposure to far-red light (FR) can reverse the effect of both R (indicating phytochrome control) and 35° C treatment. Fluence-response curves for this reversal of the effect of R and 35° C treatments are quantitatively identical. Treatment for 10 min with light of wavelenght 680, 700, 710 and 730 nm, after R and 35° C treatment, demonstrates that germination induced by 35° C treatment results from increased sensitivity to a pre-existing, active, far-red-absorbing form of phytochrome (Pfr) in the seeds.Abbreviations FR far-red light - P phytochrome - Pr red-absorbing form of P - Pfr far-red-absorbing form of P - R red light     

Immunological Cross-Reactivity Between Simian Virus 40 Large T Antigen and D2 Hybrid T Antigen

A specific antiserum was raised in rabbits against D2 hybrid T antigen that had been purified from HeLa cells infected with the adenovirus/simian virus 40 hybrid, Ad2(+)D2. The specificity of this serum was compared with that of a conventional hamster antiserum against simian virus 40-induced tumors by immunoprecipitation and by a new radioimmune assay that can detect nanogram quantities of D2 hybrid T antigen.     

Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a

The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.     

Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors

Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli.     

Comparative biological properties of a recombinant chimeric anti-carcinoma mAb and a recombinant aglycosylated variant

Summary It has been demonstrated previously that the degree of glycosylation of a molecule may alter its pharmacokinetic properties and, in the case of an antibody, its metabolism and other biological properties. Transfectomas producing aglycosylated chimeric B72.3(1) pancarcinoma monoclonal antibody (mAb) were developed by introduction of the eukaryotic expression construct pECMgpB72.3 HuG1-agly, into SP2/0 murine myeloma cells producing the chimeric chain of mAb B72.3. After cell cloning, one subclone with the highest binding to the TAG-72-positive human colon carcinoma was designated mAb aGcB72.3, and its biological and biochemical properties were compared with those of the chimeric B72.3(1), designated mAb cB72.3. Polyacrylamide gel electrophoresis showed that under non-reducing conditions, the molecular masses of the aGcB72.3 and cB72.3 mAbs were 162 kDa and 166 kDa respectively. The heavy chain of mAb aGcB72.3 had a slightly faster mobility than that of cB72.3, while the mobility of the light chains of the two chimeric mAbs was similar. No difference was observed in the isoelectric points of either chimeric mAb. Liquid competition radioimmunoassays demonstrated that the aGcB72.3 and cB72.3 mAbs have comparable binding properties to TAG-72. These studies demonstrate that aglycosylation of the chimeric IgG1 mAb B72.3 at theCh2 domain, as has been shown for other mAbs [Dorai H., Mueller B., Reisfeld R. A., Gillies S. D. (1991) Hybridoma 10:211; Morrison S. L., Oi V. T. (1989) Adv Immunol 44:65], eliminates antibody-dependent cell-mediated cytotoxicity activity, but does not substantially alter affinity or plasma clearance in mice. These studies also demonstrate for the first time (a) no difference in plasma clearance of an aglycosylated and a chimeric mAb in a primate after i.v. inoculation; (b) a difference (P 0.05) in mice in the more rapid peritoneal clearance of a chimeric mAb versus an aglycosylated chimeric mAb; (c) higher (0.05 P 0.1) tumor:liver ratios at 24, 72 and 168 h using¹¹¹In-labeled aglycosylated chimeric mAb versus chimeric mAb. Since the liver is the major site of metastatic spread for most carcinomas, slight differences in tumor to normal liver ratios may be important in diagnostic applications. These studies thus indicate that comparative analyses of a novel recombinant construct (i.e., aglycosylated) and its standard chimeric counterpart require documentation in more than one system and are necessary if one is ultimately to define optimal recombinant/chimeric constructs for diagnosis and therapy in humans.     

蓖麻蚕两种凝集素在蚕体内的出现与分布

蓖麻蚕Philosamia cynthia ricni血淋巴含两种凝集素,一种凝集兔新鲜红血球,凝血活力被L-鼠李糖和D-半乳糖抑制;另一种凝集戊二醛固定的人和鸡的红血球,凝血活力被岩藻糖抑制.它们在蚕的不同生长阶段及在蚕体各组织中的分布和凝血活力显著不同.血淋巴中这两种凝集素的凝血活力明显比其他组织中高.卵中测不到这两种凝集素活力.本文对这两种凝集素在蚕体中可能的生理功能进行了讨论.