One hundred years of zygotic embryo culture investigations

Summary Isolation of zygotic embryos from seeds and their culture in a defined medium, initiated by Hanning in 1904, has proved to be a promising method to study the factors that control growth and differentiation of embryos. Using this technique, several investigations have focused on the carbohydrate and nitrogen nutrition during germination of cultured seed embryos and on the effects of plant hormones on their morphogenesis. Culture of immature embryos leads to their germination into weak seeldings, skipping the later stages of embryogenesis, by a process known as precocious germination. Progressively smaller embryos have been cultured by supplementation of the medium with coconut milk or hormonal additives or by osmotic adjustment of the medium by high concentrations of sucrose or mannitol. Although methods have not been developed for large-scale isolation and culture of zygotes, zygotes of maize isolated from embryo sacs and those obtained by in vitro fertilization have been grown in culture into full-term embryos. Embryo culture techniques are widely used to rescue embryos from seed of wide crosses which usually abort and to overcome dormancy of recalcitrant seeds.     

An N-Terminal Domain of Herpes Simplex Virus Type I gE Is Capable of Forming Stable Complexes with gI

Using limited proteolytic analyses, we show that gE present in soluble herpes simplex virus type 1 gE-gI complexes is cleaved into a C-terminal (CgE) and an N-terminal (NgE) domain. The domain boundary is in the vicinity of residue 188 of mature gE. NgE, but not CgE, forms a stable complex with soluble gI.     

A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis

The Snm secretion system is a crucial virulence determinant of Mycobacterium tuberculosis. Genes encoding all known components of this alternative secretion pathway are clustered at the same genetic locus, known as RD1. Here, we show that a mutant M. tuberculosis strain containing a transposon insertion in the Rv3615c gene, which is situated outside the RD1 locus, results in loss of Snm secretion. Complementation analysis revealed that both Rv3615c and the downstream gene Rv3614c are required for Snm secretion. Thus, we have renamed the two genes snm9 and snm10 respectively. The snm9::Tn mutant phenocopies bona fide snm mutants, exhibiting attenuation in mice, macrophage growth defects and failure to suppress cytokine induction. Furthermore, yeast two-hybrid analysis revealed a physical interaction between Snm10 and Snm7 (Rv3882c), suggesting that Snm10 may function in complex with other Snm proteins during secretion. Thus, snm9 and snm10 are the first genes located outside the RD1 locus identified as critical components of Snm secretion. These data indicate that Snm secretion consists of an elaborate network of interactions that likely arose from multiple duplication events during the evolution of M. tuberculosis.     

Anesthetic preconditioning: triggering role of reactive oxygen and nitrogen species in isolated hearts

We postulated that anesthetic preconditioning (APC) is triggered by reactive oxygen/nitrogen species (ROS/RNS). We used the isolated guinea pig heart perfused with L-tyrosine, which reacts with ROS and RNS to form strong oxidants, principally peroxynitrite (ONOO(-)), and then forms fluorescent dityrosine. ROS scavengers superoxide dismutase, catalase, and glutathione (SCG) and NO. synthesis inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) were given 5 min before and after sevoflurane preconditioning stimuli. Drugs were washed out before 30 min of ischemia and 120 min of reperfusion. Groups were control (nontreated ischemia control), APC (two, 2-min periods of perfusion with 0.32 +/- 0.02 mM of sevoflurane; separated by a 6-min period of perfusion without sevoflurane), SCG, APC + SCG, L-NAME, and APC + L-NAME. Effluent dityrosine at 1 min reperfusion was 56 +/- 6 (SE), 15 +/- 5, 40 +/- 5(++), 39 +/- 4(++), 35 +/- 4(++) , and 33 +/- 5(++) units ((++)P< 0.05 vs. APC), respectively; left ventricular pressure (%baseline) at 60 min of reperfusion was 30 +/- 5(++), 60 +/- 4, 35 +/- 5(++), 37 +/- 5(++), 44 +/- 4, and 47 +/- 4; and infarct size (%total heart weight) was 50 +/- 5(++), 19 +/- 2, 48 +/- 3(++), 46 +/- 4(++), 42 +/- 4(++), and 45 +/- 2(++). Thus APC is initiated by ROS as shown by improved function, reduced infarct size, and reduced dityrosine on reperfusion; protective and ROS/RNS-reducing effect of APC were attenuated when bracketed by ROS scavengers or NO* inhibition.     

Role of glycine-33 and methionine-35 in Alzheimer's amyloid beta-peptide 1-42-associated oxidative stress and neurotoxicity

Recent theoretical calculations predicted that Gly33 of one molecule of amyloid beta-peptide (1-42) (Abeta(1-42)) is attacked by a putative sulfur-based free radical of methionine residue 35 of an adjacent peptide. This would lead to a carbon-centered free radical on Gly33 that would immediately bind oxygen to form a peroxyl free radical. Such peroxyl free radicals could contribute to the reported Abeta(1-42)-induced lipid peroxidation, protein oxidation, and neurotoxicity, all of which are prevented by the chain-breaking antioxidant vitamin E. In the theoretical calculations, it was shown that no other amino acid, only Gly, could undergo such a reaction. To test this prediction we studied the effects of substitution of Gly33 of Abeta(1-42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Abeta(1-42) was substituted by Val (Abeta(1-42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Abeta(1-42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Abeta(1-42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence - a measure of reactive oxygen species (ROS) - in comparison to native Abeta(1-42) treatment. The ability of the peptides to generate ROS was also evaluated by electron paramagnetic resonance (EPR) spin trapping methods using the ultrapure spin trap N-tert-butyl-alpha-phenylnitrone (PBN). While Abeta(1-42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Abeta(1-42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Abeta(1-42G33V) does not form fibrils nearly as well as Abeta(1-42) after 48 h of incubation. The results suggest that Gly33 may be a possible site of free radical propagation processes that are initiated on Met35 of Abeta(1-42) and that contribute to the peptide's toxicity in Alzheimer's disease brain.     

Derivatives of xanthic acid are novel antioxidants: application to synaptosomes

Xanthic acids have long been known to act as reducing agents. Recently, D609, a tricyclodecanol derivative of xanthic acid, has been reported to have anti-apoptotic and anti-inflammatory properties that are attributed to specific inhibition of phosphatidyl choline phospholipase C (PC-PLC). However, because oxidative stress is involved in both of these cellular responses, the possibility that xanthates may act as antioxidants was investigated in the current study. Finding that xanthates efficiently scavenge hydroxyl radicals, the mechanism by which D609 and other xanthate derivatives may protect against oxidative damage was further examined. The xanthates studied, especially D609, mimic glutathione (GSH). Xanthates scavenge hydroxyl radicals and hydrogen peroxide, form disulfide bonds (dixanthogens), and react with electrophilic products of lipid oxidation (acrolein) in a manner similar to GSH. Further, upon disulfide formation, dixanthogens are reduced by glutathione reductase to a redox active xanthate. Supporting its role as an antioxidant, D609 significantly (p < 0.01) reduces free radical-induced changes in synaptosomal lipid peroxidation (TBARs), protein oxidation (protein carbonyls), and protein conformation. Thus, in addition to inhibitory effects on PC-PLC, D609 may prevent cellular apoptotic and inflammatory cascades by acting as antioxidants and novel GSH mimics. These results are discussed with reference to potential therapeutic application of D609 in oxidative stress conditions.     

Role of ascorbate in the regulation of nitric oxide generation by polymorphonuclear leukocytes

We have recently demonstrated that NO-mediated polymorphonuclear (PMN)-dependent inhibition of rat platelet aggregation is significantly enhanced in the presence of ascorbate. Consequently, the present study was undertaken to elucidate the underlying mechanisms involved in ascorbate-mediated potentiation of NO synthesis in PMNs. We observed that ascorbate or its oxidized product, dehydroascorbate (DHA), enhanced NOS activity, as measured by nitrite content, diaminofluorescein fluorescence or conversion of L-[3H]arginine to L-[3H]citrulline in rat, monkey, and human PMNs. The increase in NO generation following ascorbate treatment was due to the intracellular ascorbate as iodoacetamide-mediated inhibition of DHA to ascorbate conversion attenuated the DHA-mediated increase in NO synthesis. The augmentation of NOS activity in the PMN homogenate by tetrahydrobiopterin was significantly enhanced by ascorbate, while ascorbate alone did not influence the NOS activity. Ascorbate-mediated enhancement of NOS activity in the cultured PMNs was significantly reduced in the presence of biopterin synthesis inhibitors. Ascorbate, thus, seems to regulate the NOS activity in the PMNs through tetrahydrobiopterin.     

Protein minimization of the gp120 binding region of human CD4

CD4 is an important component of the immune system and is also the cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. Constructs consisting of all four extracellular domains of human CD4 as well as the first two domains (CD4D12) have previously been expressed and characterized. All of the gp120-binding residues are located within the first N-terminal domain (D1) of CD4. To date, it has not been possible to obtain domain D1 alone in a soluble and active form. Most residues in CD4 that interact with gp120 lie within the region 21-64 of domain D1 of CD4. On the basis of these observations and analysis of the crystal structure of CD4D12, a mutational strategy was designed to express CD4D1 and region 21-64 of CD4 (CD4PEP1) in Escherichia coli. K(D) values for the binding of CD4 analogues described above to gp120 were measured using a Biacore-based solution-phase competition binding assay. Measured K(D) values were 15 nM, 40 nM, and 26 microM for CD4D12, CD4D1, and CD4PEP1, respectively. All of the proteins interact with gp120 and are able to expose the 17b-binding epitope of gp120. Structural content was determined using CD and proteolysis. Both CD4D1 and CD4PEP1 were partially structured and showed an enhanced structure in the presence of the osmolyte sarcosine. The aggregation behavior of all of the proteins was characterized. While CD4D1 and CD4PEP1 did not aggregate, CD4D12 formed amyloid fibrils at neutral pH within a week at 278 K. These CD4 derivatives should be useful tools in HIV vaccine design and entry inhibition studies.