马尾树科及其近缘科花粉形态研究

本文观察了马尾树科(Rhoipteleaceae)、胡桃科(Juglandaceae)以及桦木科(Be-tulaceae)、9属9种植物花粉形态。马尾树科与桦木科桦木属(Betula L.)花粉粒均为扁球形,极面观呈钝三角形,萌发孔类型均为多孔类型,孔之间均有弓形加厚,而且外表面均具细颗粒状雕纹。而胡桃科大多为多边形、散孔类型。根据花粉形态资料,探讨了马尾树科的系统位置。     

The effect of berbamine derivatives on activated Ca2+-stimulated Mg2+-dependent ATPase in erythrocyte membranes.

Ca2+-stimulated Mg2+-dependent ATPase (Ca2+ + Mg2+-ATPase) stimulated by calmodulin, by partial proteolysis or by oleic acid in erythrocyte membranes was inhibited by various derivatives of the naturally occurring alkaloid berbamine. The ability of these derivatives to inhibit trypsin-activated Ca2+ + Mg2+-ATPase correlated well with their ability to inhibit the calmodulin-stimulated enzyme. Inhibition of the trypsin-activated Ca2+ + Mg2+-ATPase by O-4-(ethoxybutyl)berbamine (EBB) was competitive with respect to ATP. The Ki for inhibition was about 8 microM. These results suggest that the binding site of EBB on the activated Ca2+ + Mg2+-ATPase may bear structural similarity to that on calmodulin, and may be closely related to the ATP-binding site on the enzyme.     

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

Comparative EPR studies on the nitrite reductases from Escherichia coli and Wolinella succinogenes

Hexaheme nitrite reductases purified to homogeneity from Escherichia coli K-12 and Wolinella succinogenes were studied by low-temperature EPR spectroscopy. In their isolated states, the two enzymes revealed nearly identical EPR spectra when measured at 12 K. Both high-spin and low-spin ferric heme EPR resonances with g values of 9.7, 3.7, 2.9, 2.3 and 1.5 were observed. These signals disappeared upon reduction by dithionite. Reaction of reduced enzyme with nitrite resulted in the formation of ferrous heme-NO complexes with distinct EPR spectral characteristics. The heme-NO complexes formed with the two enzymes differed, however, in g values and line-shapes. When reacted with hydroxylamine, reduced enzymes also showed the formation of ferrous heme-NO complexes. These results suggested the involvement of an enzyme-bound NO intermediate during the six-electron reduction of nitrite to ammonia catalyzed by these two hexaheme nitrite reductases. Heme proteins that can either expose bound NO to reduction or release it are significant components of both assimilatory and dissimilatory metabolisms of nitrate. The different ferrous heme-NO complexes detected for the two enzymes indicated, nevertheless, their subtle variation in heme reactivity during the reduction reaction.     

Structural analysis of the locus containing the human C-reactive protein gene and its related pseudogene

The gene for human C-reactive protein (CRP) is mapped within a 34-kilobase pair genomic DNA segment identified by chromosome walking through overlapping DNA fragments cloned into a lambda phage library. Within 16 kilobase pairs upstream and downstream of the locus for the authentic CRP gene, only one other sequence homologous to that for CRP could be found. Sequencing analysis indicates this sequence to be a pseudogene with 50-80% region-specific homology. Comparison of the authentic CRP gene cloned from genomic DNA libraries independently prepared from three patients indicates no difference in the 5' and 3' flanking region, promoter region, or coding sequence. Only a polymorphism in the length of the poly(GT) stretch located in the intron is observed. There appears to be only one gene locus and copy per haploid chromosome for the authentic CRP gene and its pseudogene.     

Reactivation of inhibited bone acid phosphatase and its significance in bone histomorphometry

Despite biochemical demonstration of acid phosphatase (AcP) activation or reactivation in bone, few attempts have been made to show similar effects histochemically. Bones from growing rats, when fixed in 4% buffered formaldehyde at room temperature and demineralized in 5% formic acid, exhibited expected inactivation of AcP. The inhibited AcP, however, was reactivated by pre-incubation of sections for 1 hr at 37 degrees C in the following buffers: 0.2 M Tris, 0.2 M glycine, 0.2 M NaHCO3, or 0.1 M borax, as well as in alkaline water, but not in 0.2 M Na2HPO4 (all at pH 9). The reactivation was (a) site-specific (e.g., osteoclasts, osteoblasts, osteocytes, and cement lines), (b) temperature- and pH-dependent, (c) unaffected by OH- or SH--binding agents or by an alkaline phosphatase inhibitor, and (d) inhibited completely by 10 mM Na2HPO4. The reactivation process, much simplified and/or more effective than with the methods previously reported, was observed in all 83 human biopsy bones embedded in methyl methacrylate and in human bones stored in cold buffered formaldehyde for 7 months. This study demonstrates a unique method for reactivating and thus localizing the inhibited AcP in bones, and suggests possible applications in bone histomorphometry.     

Isolation and characterization of human VkIII germ-line genes. Implications for the molecular basis of human VkIII light chain diversity

To understand the relative importance of germ-line genes in the generation of the functional human antibody repertoire, it is first necessary to define the number of variable region genes and to determine their fine structure. We have focused on the human VkIII variable region gene family because of its association with autoantibodies. A human genomic library was screened with a VkIII cDNA probe and subsequently with a VkIII germ-line gene probe. Seven different VkIII clones were isolated and characterized by restriction mapping and sequence analyses. Three clones have identical restriction enzyme sites over a 12-kilobase (kb) region, contain identical sequences over an 895-base pair (bp) region, and thus are likely to be different isolates of the same human VkIII gene. Another two clones have identical restriction enzyme sites over a 5-kb region, are identical over a stretch of 905 bp sequenced, and likely represent independent isolates of another human VkIII gene. The remaining two VkIII clones consist of two additional VkIII genes which are homologous to each other, but are quite different from the first two VkIII genes. Thus, four new human VkIII genes were defined. Together with four other VkIII genes previously isolated by other investigators, a total of eight human VkIII germ-like genes have now been described. A comparison of the deduced amino acid sequences of these genes with the reported amino acid sequences of all human VkIII light chains suggests that at least one additional VkIII gene exists in the germ line. Among the eight identified human germ-line VkIII genes, three are pseudogenes. Of the remaining five potential functional genes, one gene seems to encode a majority of the VkIII light chains which have been sequenced. Possible explanations for this phenomenon are discussed.     

Immune regulation of the L5178Y murine tumor-dormant state. II. Interferon-gamma requires tumor necrosis factor to restrain tumor cell growth in peritoneal cell cultures from tumor-dormant mice

L5178Y lymphoma cells are restrained from progressive growth in peritoneal cell ("in vitro tumor-regressor" PC) cultures prepared from many DBA/2 mice which harbor the tumor cells in the peritoneal cavity in a tumor-dormant state. Treatment of these PC cultures with 'antibodies to murine interferon-gamma (MuIFN-gamma) and murine tumor necrosis factor (MuTNF) but not with antibody to interleukin 2 (IL-2) receptors eliminated the restraint on tumor cell growth and permitted their progressive proliferation. L5178Y cells were found to be resistant to the direct toxic effects of large concentrations (3,000 U/ml) of MuIFN-gamma and of MuTNF, either alone or in combination. Treatment of PC cultures from tumor-dormant mice, in which tumor cells grew progressively ("in vitro tumor-progressor"), but not PC cultures from normal mice, with exogenous MuIFN-gamma resulted in a marked inhibition of tumor cell growth. The MuIFN-gamma-induced cytotoxic activity was cell-mediated since no soluble tumor-cytotoxic factors could be detected in the cultures. MuIFN-gamma induced cytotoxic activity in plastic-adherent peritoneal cell (AD-PC) cultures, but induced no cytotoxic activity in nonadherent-PC cultures unless small numbers (2%) of AD-PC were present, and inclusion of antibody to MuTNF in these mixed PC cultures blocked the development of cytotoxic activity. Antibody to MuTNF also blocked the development of cytotoxic activity in cultures of MuIFN-gamma-treated whole PC and AD-PC from tumor-dormant mice. These results indicate that MuIFN-gamma and MuTNF are both important in restraining tumor cell growth in PC cultures from tumor-dormant mice, and that MuIFN-gamma requires the presence of MuTNF to induce cytotoxic activity in these cultures.     

Expression of a recombinant murine IgE in transfected myeloma cells

We constructed a recombinant gene encoding an immunoglobulin (Ig) heavy chain consisting of the variable region from the phosphorylcholine (PC)-specific secreting myeloma MOPC167 and the epsilon constant region from SJL mice. This gene, cloned into the shuttle vector pSV2gpt, was transfected into J558L myeloma cells, and stable transformants that expressed the epsilon gene were cloned. The IgE heavy chain in these transformants is associated with the endogenous lambda light chain and is secreted as an intact IgE molecule. However, the secreted IgE does not bind to PC conjugated to bovine serum albumin (PC-BSA). The MOPC167 kappa chain gene was cloned into the shuttle vector pSV2neo and was transfected into the epsilon heavy-chain transformant. Stable transformants were cloned that expressed both the epsilon heavy chain and the kappa light chain. IgE secreted from such a transformant was shown to bind to PC-BSA. Both types of secreted recombinant IgE bound to rat basophilic leukemia (RBL) cells, but only the IgE produced by the cell line transformed with the MOPC167 kappa gene could be cross-linked with PC-BSA to cause serotonin release.