Cellular Networks in Epidermal Peels of Onion

Cellular networks ill epidermal peels of onion bulb can be distinguished by first removal of superficially attached cytoplasmic constituents with Triton phos- phate buffer and then by staining with Coomassie blue R 250(Fig. 4). Two distinct kinds of networks can be further recognized by treatment with colchicine and cytochalasins: one thinner network underneath the periphery of plasmalemma can be abolished by colchicine (Fig. 7, 8); and the other thicker one which associates tangentially with the nucleus was more distinctive after cytochalasin B treatment (Fig. 5, 6). Discussion is made regarding these two networks in wall-enclosed plant cells as revealed by the present technic.     

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

Herpes simplex virus (HSV) entry into cells requires four membrane glycoproteins: gD is the receptor binding protein, and gB and gH/gL constitute the core fusion machinery. Crystal structures of gD and its receptors have provided a basis for understanding the initial triggering steps, but how the core fusion proteins function remains unknown. The gB crystal structure shows that it is a class III fusion protein, yet unlike other class members, gB itself does not cause fusion. Bimolecular complementation (BiMC) studies have shown that gD-receptor binding triggers an interaction between gB and gH/gL and concurrently triggers fusion. Left unanswered was whether BiMC led to fusion or was a by-product of it. We used gB monoclonal antibodies (MAbs) to block different aspects of these events. Non-virus-neutralizing MAbs to gB failed to block BiMC or fusion. In contrast, gB MAbs that neutralize virus blocked fusion. These MAbs map to three functional regions (FR) of gB. MAbs to FR1, which contains the fusion loops, and FR2 blocked both BiMC and fusion. In contrast, MAbs to FR3, a region involved in receptor binding, blocked fusion but not BiMC. Thus, FR3 MAbs separate the BiMC interaction from fusion, suggesting that BiMC occurs prior to fusion. When substituted for wild-type (wt) gB, fusion loop mutants blocked fusion and BiMC, suggesting that loop insertion precedes BiMC. Thus, we postulate that each of the gB FRs are involved in different aspects of the path leading to fusion. Upon triggering by gD, gB fusion loops are inserted into target lipid membranes. gB then interacts with gH/gL, and this interaction is eventually followed by fusion.Entry of herpes simplex virus (HSV) into cells requires four viral glycoproteins, gB, gD, gH, and gL, plus one of several cell receptors, either herpesvirus entry mediator (HVEM), nectin-1, or 3-OST (45). Crystal structures and other studies have documented that receptor binding triggers conformational changes to gD that trigger the downstream events leading to fusion (10, 11, 18, 26, 28, 52). Moreover, when HSV receptor-bearing cells are transfected with expression plasmids for glycoproteins gB, gD, gH, and gL, the cells fuse to form multinucleated giant cells or syncytia (39, 48). However, the precise series of events that take place after receptor binding have not yet been fully elucidated. What we do know is that both gB and a heterodimer of gH/gL constitute the core fusion machinery that is conserved and required for the fusion step of entry of all herpesviruses (18, 26, 30, 46, 49).Thus far, we know the crystal structure of one form of the gB ectodomain of HSV type 1 (HSV-1) (19). This protein has the characteristics of a fusion protein and is a charter member of the class III group of viral fusion proteins (4). Others in this class include Epstein-Barr virus gB, vesicular stomatitis virus (VSV) G, and baculovirus gp64 (5, 22, 41). Like VSV G and gp64, gB has two putative fusion loops at the base of each protomer of the crystallized trimer. Single-amino-acid mutations in many of the hydrophobic residues of the putative fusion loops of gB ablate its ability to function in cell-cell fusion assays (16, 17). Moreover, these mutants are unable to complement the entry of a gB-null virus (16). Finally, the ectodomains of these mutants, unlike wild-type protein, failed to coassociate with liposomes, indicating that the putative fusion loops do insert into membranes (16, 17). Recently, it was shown that several of these mutants are also defective for fusion events involved in virus egress (51). Together, these studies provide compelling evidence that HSV gB functions as a fusion protein and that the fusion loops are critical for this function. However, unlike VSV G and baculovirus gp64, gB does not function on its own in entry but, rather, requires the participation of gH/gL. In the absence of crystallographic data for gH/gL, it is not yet clear what role it plays in herpesvirus fusion. In a previous study, we used bimolecular complementation (BiMC) to examine protein-protein interactions that occur among the viral glycoproteins during fusion (1). A similar study was carried out by Avitabile et al. (2). The BiMC assay is based on the observation that N- and C-terminal fragments of green fluorescent protein (GFP) (and derivatives such as enhanced yellow fluorescent protein [EYFP]) do not spontaneously reconstitute a functional fluorophore (20, 29, 40). However, the codons for each half can be appended to the genes for two interacting proteins (23, 24). When these are cotransfected, an interaction between the two proteins of interest brings the two halves of the fluorophore in close enough contact to restore fluorescence.When HSV receptor-bearing cells, such as B78H1 cells that are engineered to express nectin-1, are transfected with plasmids that express gB, gD, gH, and gL, they undergo cell-cell fusion (13, 15, 27, 31, 48). When gD is omitted, no fusion occurs. We found that fusion of these transfected cells could be triggered by addition of a soluble form of gD (the gD ectodomain). We then used this approach to examine interactions between gB and gH/gL during cell fusion (1). Therefore, we tagged gB with the C-terminal half of EYFP and gH with the N-terminal half. When plasmids bearing these forms were cotransfected into C10 cells along with a plasmid for untagged gL, no fusion occurred, but importantly, no BiMC occurred. However, when we added gD306, cells began to fuse within 10 min, and all of the syncytia that formed exhibited bright EYFP fluorescence indicative of BiMC. We concluded that gD triggers both fusion and a physical interaction between gB and gH/gL. However, these experiments did not separate these two events, so we were unable to determine if the interaction preceded fusion or merely was a by-product of it.The purpose of this study was to determine if the gB-gH/gL interaction is essential for fusion and if it occurs prior to fusion. We focused on gB because its structure is known and we have a panel of well-characterized monoclonal antibodies (MAbs) to gB. Our approach was to determine which of these MAbs, if any, could block fusion and also block the interaction with gH/gL. We also examined the effect of mutations to the fusion loops of gB on its interaction with gH/gL. We previously mapped these MAbs to four functional regions (FR) of gB, three of which were resolved in the crystal structure (6, 19). Of these, FR1 contains the fusion loops, FR2 is in the center of the gB structure with no known function, and FR3 is at in the crown of the protein and may be involved in binding to cells (7). Our rationale was that if the interaction between gB and gH/gL is important for fusion, then it should not be blocked by nonneutralizing anti-gB MAbs. At the same time, we thought that some neutralizing MAbs might not only block fusion but also block BiMC. We found that neutralizing MAbs to FR1 and FR2 inhibited both BiMC and fusion. In contrast, we found that neutralizing MAbs that map to FR3 blocked fusion but failed to block the interaction between gB and gH/gL, thereby dissociating the two events. Finally, we found that gB mutants with changes in the fusion loops that were fusion negative were also unable to bind to gH/gL. The latter results suggest that insertion of gB into the target membrane precedes its interaction with gH/gL.     

A residue in MutY important for catalysis identified by photocross-linking and mass spectrometry

MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. MutY contains a [4Fe-4S]2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif. This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily. Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY. The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY. The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry. This analysis revealed Arg 143 as the site of modification in MutY. Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily. Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs. In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G. Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone. The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the [4Fe-4S]2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate.     

Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor

Cerebrovascular deposition of amyloid beta-protein (Abeta) is a common pathological feature of Alzheimer's disease and related disorders. In particular, the Dutch E22Q and Iowa D23N mutations in Abeta cause familial cerebrovascular amyloidosis with abundant diffuse amyloid plaque deposits. Both of these charge-altering mutations enhance the fibrillogenic and pathogenic properties of Abeta in vitro. Here, we describe the generation of several transgenic mouse lines (Tg-SwDI) expressing human neuronal Abeta precursor protein (AbetaPP) harboring the Swedish K670N/M671L and vasculotropic Dutch/Iowa E693Q/D694N mutations under the control of the mouse Thy1.2 promoter. Tg-SwDI mice expressed transgenic human AbetaPP only in the brain, but at levels below those of endogenous mouse AbetaPP. Despite the paucity of human AbetaPP expression, quantitative enzyme-linked immunosorbent assay measurements revealed that Tg-SwDI mice developed early-onset and robust accumulation of Abeta in the brain with high association with isolated cerebral microvessels. Tg-SwDI mice exhibited striking perivascular/vascular Abeta deposits that markedly increased with age. The vascular Abeta accumulations were fibrillar, exhibiting strong thioflavin S staining, and occasionally presented signs of microhemorrhage. In addition, numerous largely diffuse, plaque-like structures were observed starting at 3 months of age. In vivo transport studies demonstrated that Dutch/Iowa mutant Abeta was more readily retained in the brain compared with wild-type Abeta. These results with Tg-SwDI mice demonstrate that overexpression of human AbetaPP is not required for early-onset and robust accumulation of both vascular and parenchymal Abeta in mouse brain.     

Partitioning diversity for conservation analyses

Aim  Differentiation of sites or communities is often measured by partitioning regional or gamma diversity into additive or multiplicative alpha and beta components. The beta component and the ratio of within-group to total diversity (alpha/gamma) are then used to infer the compositional differentiation or similarity of the sites. There is debate about the appropriate measures and partitioning formulas for this purpose. We test the main partitioning methods, using empirical and simulated data, to see if some of these methods lead to false conclusions, and we show how to resolve the problems that we uncover.
Location  South America, Ecuador, Orellana province, Rio Shiripuno.
Methods  We construct sets of real and simulated tropical butterfly communities that can be unambiguously ranked according to their degree of differentiation. We then test whether beta and similarity measures from the different partitioning approaches rank these datasets correctly.
Results  The ratio of within-group diversity to total diversity does not reflect compositional similarity, when the Gini–Simpson index or Shannon entropy are used to measure diversity. Additive beta diversity based on the Gini–Simpson index does not reflect the degree of differentiation between N sites or communities.
Main conclusions  The ratio of within-group to total diversity (alpha/gamma) should not be used to measure the compositional similarity of groups, if diversity is equated with Shannon entropy or the Gini–Simpson index. Conversion of these measures to effective number of species solves these problems. Additive Gini–Simpson beta diversity does not directly reflect the differentiation of N samples or communities. However, when properly transformed onto the unit interval so as to remove the dependence on alpha and N , additive and multiplicative beta measures yield identical normalized measures of relative similarity and differentiation.     

Development of W/O Microemulsion for Transdermal Delivery of Iodide Ions

The objective of this study was to develop a water-in-oil (w/o) microemulsion which can be utilized as a transdermal delivery for iodide ions. Several w/o microemulsion formulations were prepared utilizing Span 20, ethanol, Capryol 90®, and water. The selected formulations had 5%, 10%, 15%, 20%, and a maximum of 23% w/w water content. Potassium iodide (KI) was incorporated in all formulations at 5% w/v. Physicochemical characterizations were conducted to evaluate the structure and stability. These studies included: mean droplet size, pH, viscosity, conductivity, and chemical stability tests. In vitro human skin permeation studies were conducted to evaluate the diffusion of the iodide ion through human skin. The w/o microemulsion formulations were stable and compatible with iodide ions with water content ranging from 5% to 23% w/w. The addition of KI influenced the physicochemical properties of microemulsion as compared to blank microemulsion formulations. In vitro human skin permeation studies indicated that selected formulations improved iodide ion diffusion significantly as compared to control (KI solution; P value < 0.05). Iodide ions were entrapped within the aqueous core of w/o microemulsion. Span 20, ethanol and Capryol 90 protected the iodide ions against oxidation and formed a stable microemulsion. It is worth to note that according to Hofmeister series, iodide ions tend to lower the interfacial tension between water and oil and consequently enhance overall stability. This work illustrates that microemulsion system can be utilized as a vehicle for the transdermal administration of iodide.KEY WORDS: iodide, microemulsion, skin permeation, transdermal     

无创性肢体缺血预适应对小鼠抗应激能力的影响

目的:探讨无创性肢体缺血预适应在提高小鼠抗应激能力方面的作用。方法:小鼠分为正常组、对照组、预适应组和药物组,进行常压耐缺氧、耐疲劳负重游泳、耐高、低温实验,分别记录各种应激状态下小鼠的耐受时间,测定常压缺氧耐受后小鼠血清中SOD的活性和负重游泳后血清中乳酸的含量。结果:预适应组能显著提高小鼠常压耐缺氧时间,且SOD活性较对照组有显著的提高,但提高的程度均低于普萘洛尔组。预适应组平均游泳时间显著延长,且程度与咖啡因组相当。预适应能明显延长高温下小鼠的存活时间,且延长程度与氯丙嗪没有显著性差异。与正常组比较,预适应组能显著延长小鼠的耐低温时间。结论:无创性肢体缺血预适应能提高小鼠耐缺氧、抗疲劳、耐高温和耐低温的能力。     

Regeneration of doubled haploid plants by androgenesis of cucumber (<Emphasis Type="Italic">Cucumis sativus </Emphasis>L.)

Six experiments (including pretreatment, embryonic callus induction media, preculture conditions, embryo induction media, embryo germination media, and genotypic effects) were conducted to develop an efficient cucumber (Cucumis sativus L., 2n = 2x = 14) anther culture protocol. Pretreatment and embryo induction were key factors for successful anther culture. Suitable temperature stress depended on the ecotype, i.e., cucumbers from cold areas responded well to cold shock whereas those from temperate areas responded well to heat treatment. The best medium for embryonic callus induction was MS medium supplemented with 4.44 μM BA, 2.26 μM 2, 4-D, 4.64 μM KIN, 3% sucrose and 0.8% agar. For embryo induction, MS medium supplemented with 0.54 μM NAA, 13.32 μM BA, 3% sucrose and 0.8% agar was optimal, and for embryo germination MS medium containing 2.22 μM BA, 6% sucrose and 1.2% agar was best. Using this protocol, we produced callus from 16 genotypes and regenerated plants from three of 20 evaluated. Three embryos per anther and 42 DH per 45 anthers (93% success) were obtained for cv. Ningjia No. 1, which was an improved result over a previous report. The origin of regenerants from microspores was determined by cytological, morphological and AFLP analyses.     

Prokaryotic expression and polyclonal antibody preparation of a novel Rab-like protein mRabL5

Rab GTPases, which belong to the Ras superfamily, represent a group of small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. We first identified mRabL5 (GenBank Accession No. NP_080349), a novel Mus musculus Rab-like protein, present as a Golgi-associated protein. Here we presented the results of the cloning, prokaryotic expression, purification, and polyclonal antibody production of the novel Rab-like protein. In order to obtain a specific antibody against mRabL5, we prepared two GST fusion proteins, full-length mRabL5 GST fusion protein and mRabL5 C terminus GST fusion protein, to immunize rabbits. Western blot analysis showed that both antibodies prepared against full length of mRabL5 and its C terminus, respectively, can recognize mRabL5 protein. Immunofluorescence of mRabL5 in NIH3T3 cells using the two antibodies showed its perinuclear clustering distribution pattern. The polyclonal antibodies preparation against mRabL5 provided a good tool for us to study the functional involvement of mRabL5.