A linkage map for CRINKLED PETAL: a homeotic gene of Clarkia tembloriensis (Onagraceae)

Homeotic mutations in flowers lead to the development of floral organs in abnormal locations. In most laboratory-induced examples of this type of mutation, two adjacent whorls of organs are affected, resulting in two whorls of abnormal organ formation. However, the crinkled petal mutant of Clarkia tembloriensis is interesting because it is a naturally occurring mutation and it affects only the second whorl of organs, producing sepaloid petals. In this study one wild-type population (Cantua Creek-2) and one crinkled petal mutant population (Red Rocks) were compared using 181 different primers in random amplified polymorphic DNA (RAPD) analysis. Bulk DNA from each parent population and their subsequent crosses were used to compare the genetic differences between the two populations and to search for molecular markers linked with the CRINKLED PETAL locus. A linkage map was developed for the CRINKLED PETAL gene, and markers were discovered which flanked both sides of the locus.     

Capturing the sublimity of a free radical gas

This paper reviews the work related to nitric oxide (NO) done by the author and his postgraduates and colleagues in the past 7 years in the National University of Singapore. Our work shows that (i) NADPH-d and NO synthase (NOS) are often but not always identical; (ii) NO (as indicated by NADPH-d histochemistry and NOS immunohistochemistry) is generated in some endocrine (thyroid, parathyroid and ultimobranchial glands) and immune (thymus and bursa of Fabricius) organs and the cochlea. It is noted from the above studies that NO could possibly regulate blood flow through the various organs via its presence in the vascular endothelial cells and also via nitrergic neurons innervating the blood vessels. It could also regulate the activity of the secretary cells of these organs by being present in them, as well as acting through nitrergic neurons closely related to them. The paper also examines the Janus-faced nature of NO as a neuroprotective and neurodestructive agent, and the apparent non-involvement of peroxynitrite and inducible NOS in neuronal death occurring in the red nucleus and nucleus dorsalis after spinal cord hemisection.     

Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623

The U.S. Environmental Protection Agency has developed method 1623 for simultaneous detection of Cryptosporidium oocysts and Giardia cysts in water. Method 1623 includes four major steps: filtration, immunomagnetic separation (IMS), fluorescent antibody (FA) staining and microscopic examination. It was noted that the recovery levels following IMS-FA and FA staining were high, averaging more than 92.0% and 89.0% for C. parvum oocysts and G. lamblia cysts, respectively. In contrast, when the filtration step was incorporated, the recovery level of C. parvum oocysts declined significantly to 18.1% in seeded tap water, while a relatively high recovery level of 77.2% for G. lamblia cysts could still be achieved. Further study indicated that the recovery level of C. parvum oocysts could be enhanced significantly when an appropriate amount of silica particles was added to a water sample. The recovery level of C. parvum oocysts was affected by particle size and concentration. The optimal silica particle size was determined to be within the range of 5-40 microm, and the corresponding optimal silica concentration was 1.42 g for 10-l tap water. When both G. lamblia cysts and C. parvum oocysts were spiked into the tap water sample containing the optimum amount of silica particles, the average recovery levels of oocysts and cysts were 82.7% and 75.4%, respectively. The results obtained clearly suggested that addition of an appropriate amount of silica particles could improve the recovery level of C. parvum oocysts significantly and yet there was no noticeable deleterious effect on the recovery level of G. lamblia cysts. Further study indicated that the rotation time in the IMS procedure using the Dynal GC-Combo IMS kit (which was recommended in method 1623) was important for G. lamblia cyst detection. In contrast, the recovery level of C. parvum oocysts was not affected by the rotation time. Furthermore, it was found that the recovery levels of C. parvum oocysts using methods 1622 and 1623 were quite close although different IMS kits were used in the two methods.     

TetraThymosinbeta is required for actin dynamics in Caenorhabditis elegans and acts via functionally different actin-binding repeats

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinbeta expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinbeta has four repeats, each related to the actin monomer-sequestering protein thymosinbeta 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinbeta binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinbeta may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinbeta is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinbeta is of crucial importance at specific developmental stages requiring actin polymerization.     

Identification of an autoinhibitory domain of p21-activated protein kinase 5

The p21-activated protein kinases (Paks) are serine/threonine protein kinases activated by binding to Rho family small GTPases, Rac and Cdc42. Recently, Pak family members have been subdivided into two groups, I and II. Group II Paks, including Pak4, Pak5, and Pak6, does not contain the highly conserved autoinhibitory domain that is found in the group I Paks members, i.e. Pak1, Pak2, and Pak3. In the present study, we have purified the glutathione S-transferase fusion form of Pak5 and shown for the first time that Pak5 autophosphorylation can be activated by GTP bound form of Cdc42. Mutation of histidine residues 19 and 22 to leucine on the p21-binding domain of Pak5 completely abolished the binding of Cdc42 and the Cdc42-mediated autophosphorylation. On the other hand, mutation of tyrosine 40 to cysteine of Cdc42 did not knockout the binding of Pak5. Analysis of C-terminal deletion mutants has identified an autoinhibitory fragment of Pak5 that is absent from other group II Pak family members. Taken together, these results suggest that Pak5, like Pak1, contains an autoinhibitory domain and its activity is regulated by Cdc42.     

Dietary components may prevent mutation-related diseases in humans

Since it is not always possible to reduce human exposure to mutagens, attempts have been directed to identify potential antimutagens and anticarcinogens for use in protecting the population against environmental disease. The purpose of this paper is to provide the reader with information about the antimutagenic and anticarcinogenic potentials of some dietary constituents and foods widely consumed in Brazil, and to reinforce diet as a key factor in determining genomic stability and preventing human diseases. In this report, we have summarized data that show interactive effects between some dietary components and specific chemical mutagens or carcinogens using in vitro and in vivo short- or medium-term assays. The summary indicates that certain dietary compounds may be useful agents for disease prevention.     

DNA-protein cross-linking via guanine oxidation: dependence upon protein and photosensitizer

DNA-protein cross-links form when guanine undergoes a 1-electron oxidation in a flash-quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA-histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a (32)P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash-quench technique as a way to generate physiologically relevant DNA-protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA.