Anti-GM3-lactam monoclonal antibodies of the IgG type recognize natural GM3-ganglioside lactone but not GM3-ganglioside

Immunization of mice with a synthetic GM₃-lactam-BSA (bovine serum albumin) conjugate (designed to emulate the corresponding natural GM₃-lactone conjugate), followed by fusion of splenocytes with myeloma cells, gave rise to more than 300 monoclonal hybridomas producing antibodies to GM₃-lactam-BSA, which did not react with Glc-BSA and BSA. Eight antibody clones were randomly chosen from the positive 300 hybridomas. The eight clones, all belonging to the IgG class, were unreactive against GM₃-ganglioside, whereas two antibodies (P5-1 and P5-3, both IgG₁, ) reacted with GM₃-ganglioside lactone. Binding of these two antibodies to the GM₃-lactam-BSA conjugate was inhibited by soluble glycosides of GM₂-, GM₃-, and GM₄-lactam and by GM₃- and GM₄-lactam, respectively, but not by Gb₃ or asialo-GM₁ and GM₂-saccharides. A third antibody (P3; IgG_2b, ) was inhibited by GM₂-, GM₃-, and GM₄-lactam, but did not recognize GM₃-ganglioside lactone.     

Microfilaments in the preprophase band of freeze substituted tobacco root cells

Summary The organization and distribution of microfilaments (MFs) in the preprophase bands (PPBs) of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) root tip cells were studied with high pressure freezing and freeze-substitution methods. MFs were present predominantly as single filaments interspersed among microtubules (MTs) throughout the PPB. Some MFs appeared to be associated with MTs, whereas others were not. This is the first time that MFs have been demonstrated in the PPB at the electron microscope level.     

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.

Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFA's effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.     

改变乙型肝炎病毒核心抗原基因3′端序列对其在原核细胞中表达的影响

乙型肝炎病毒(HBV)的核心抗原基因(C基因)编码185个氨基酸残基,在原核细胞或痘苗病毒系统中能表达并装配成27nm大小的核心抗原(HBcAg)多聚体颗粒。已证实HBV C基因3′端编码近40个氨基酸的碱基序列,不是表达形成HBcAg颗粒所必需的。用外源基因替换这部分序列,已表达出表面带有外源基因产物的杂合颗粒,它具有很好的免疫原性,成为新型的基因工程多决定簇颗粒载体疫苗。但我们的实验中发现,用另外的外源基因替换3′端序列能显著影响HBV C基因在大肠杆菌中的表达,不同组成的外源基因其影响程度有所不同。     

Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations AFP antifreeze protein - CD circular dichroism - DTT dithiothreitol - HPLC high pressure liquid chromatography - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.     

Biosynthesis, glycosylation, movement through the Golgi system, and transport to lysosomes by an N-linked carbohydrate-independent mechanism of three lysosomal integral membrane proteins

The biosynthesis, glycosylation, movement through the Golgi system, transport to lysosomes, and turnover of three lysosomal integral membrane proteins (LIMPSs) have been studied in normal rat kidney cells using specific anti-LIMP monoclonal antibodies. Immunoelectron microscopy studies revealed the presence of LIMPs in secondary lysosomes, Golgi cisterna, and coated and uncoated vesicles located in the trans-Golgi cisterna, area. Pulse-chase experiments recorded LIMP precursors of 27 (LIMP I), 72 (LIMP II), and 86 kDa (LIMP III) and mature LIMPs of 35-50 (LIMP I), 74 (LIMP II), and 90-100 kDa (LIMP III). Time course studies on the acquisition of endoglycosidase H resistance by LIMPs indicated that all three LIMPs moved from the site of their synthesis in the endoplasmic reticulum to the medial Golgi within 30-60 min after their synthesis. All three LIMPs were fully glycosylated before leaving the Golgi system, the process during which LIMP I was retained in the trans side of the organelle. LIMP I reached the lysosomes with a halftime of 2 h and LIMPs II and III with half-times of 1 h after their synthesis by a mechanism that was independent of N-linked carbohydrates. LIMPs free of N-linked carbohydrates displayed much shorter half-lives than fully glycosylated LIMPs, suggesting an important role of the sugars in protecting LIMPs against proteolytic degradation. Double immunofluorescence microscopy experiments showed that LIMP I, LIMP II, and LIMP III are localized in the same lysosomes.     

A modified method of UDS detection in vitro suitable for screening the DNA-damaging effects of chemicals

The UDS induced in cultured FL cells by exposure to chemicals was measured as hydroxyurea-resistant incorporation of 3H-TdR in the acid-insoluble fraction of the 14C-TdR-prelabelled cells synchronized by the combination of arginine starvation and pretreatment with hydroxyurea. The level of UDS is represented by the ratios of 3H/14C radioactivities which are measures of specific activities of 3H. Two direct-acting alkylating agents, MMS and MNNG, a cross-linking agent, mitomycin C, and 3 procarcinogens, B(a)P, AFB1 and cyclophosphamide elicited UDS in the absence or presence of the liver-metabolizing system. Three chemicals of unknown carcinogenicity were also able to induce UDS in this assay system, i.e., bis-(O,O-diethylphosphinothioyl)-disulphide, 4-chlorophenoxy acetic acid (sodium salt) and caramelized malt sugar. With the exception of 4-chlorophenoxy acetic acid, they were also active in the Ames test.