Inoculation of selenium hyperaccumulator Stanleya pinnata and related non‐accumulator Stanleya elata with hyperaccumulator rhizosphere fungi—investigation of effects on Se accumulation and speciation

Little is known about how fungi affect elemental accumulation in hyperaccumulators (HAs). Here, two rhizosphere fungi from selenium (Se) HA Stanleya pinnata, Alternaria seleniiphila (A1) and Aspergillus leporis (AS117), were used to inoculate S. pinnata and related non‐HA Stanleya elata. Growth and Se and sulfur (S) accumulation were analyzed. Furthermore, X‐ray microprobe analysis was used to investigate elemental distribution and speciation. Growth of S. pinnata was not affected by inoculation or by Se. Stanleya elata growth was negatively affected by AS117 and by Se, but combination of both did not reduce growth. Selenium translocation was reduced in inoculated S. pinnata, and inoculation reduced S translocation in both species. Root Se distribution and speciation were not affected by inoculation in either species; both species accumulated mainly (90%) organic Se. Sulfur, in contrast, was present equally in organic and inorganic forms in S. pinnata roots. Thus, these rhizosphere fungi can affect growth and Se and/or S accumulation, depending on host species. They generally enhanced root accumulation and reduced translocation. These effects cannot be attributed to altered plant Se speciation but may involve altered rhizosphere speciation, as these fungi are known to produce elemental Se. Reduced Se translocation may be useful in applications where toxicity to herbivores and movement of Se into the food chain is a concern. The finding that fungal inoculation can enhance root Se accumulation may be useful in Se biofortification or phytoremediation using root crop species.     

Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA:protein N-myristoyltransferase

The importance of myristoylation for the proper biological functioning of many acylated proteins has generated interest in the enzymes of the myristoylation pathway and their interactions with substrates and inhibitors. Previous observations that S-(2-oxopentadecyl)-CoA, a nonhydrolyzable methylene-bridged analogue of myristoyl-CoA, was a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT) [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1989) J. Med. Chem. 32, 1665] prompted a closer examination of the effect of substituents at the 2-position on the interactions of myristic acid and myristoyl-CoA analogues with NMT. As an initial approach, three myristic acid derivatives bearing different substituents at the 2-position, 2-fluoromyristic acid, 2-bromomyristic acid, and 2-hydroxymyristic acid, were selected for study. Both 2-bromomyristic acid and 2-hydroxymyristic acid were available commercially; 2-fluoromyristic acid was prepared synthetically. All three compounds were found to be only weak inhibitors of NMT in vitro. Of the three, 2-bromomyristic acid was the most potent (Ki = 100 microM). In cultured cells, however, 2-hydroxymyristic acid was by far the more effective inhibitor of protein myristoylation. Neither 2-hydroxymyristic acid nor 2-bromomyristic acid significantly inhibited protein palmitoylation in cultured cells, indicating that inhibition was not occurring at the level of acyl-CoA synthetase. Activation of the 2-substituted myristic acid derivatives to their corresponding acyl-CoA thioesters by acyl-CoA synthetase resulted in inhibitors of greatly increased potency. The 2-substituted acyl-CoA analogues, 2-hydroxymyristoyl-CoA, 2-bromomyristoyl-CoA, and 2-fluoromyristoyl-CoA, were synthesized and shown to be competitive inhibitors of NMT in vitro (Ki's = 45, 450, and 200 nM, respectively). These data suggested that the enhanced inhibitory potency of 2-hydroxymyristic acid seen in cells was most probably a result of its metabolic activation to the CoA thioester. The presence of substituents at the 2-position also affected the ability of the acyl group to be transferred by NMT to a peptide substrate. Of the three acyl-CoA analogues, only 2-fluoromyristoyl-CoA served as a substrate for NMT.     

Structural and posttranslational analysis of human calcium-binding protein,spermatid-associated 1

Recently, we detected a novel biomarker in human saliva called calcium-binding protein, spermatid-associated 1 (CABS1). CABS1 protein had previously been described only in testis, and little was known of its characteristics other than it was considered a structurally disordered protein. Levels of human CABS1 (hCABS1) in saliva correlate with stress, whereas smaller sized forms of hCABS1 in saliva are associated with resilience to stress. Interestingly, hCABS1 also has an anti-inflammatory peptide sequence near its carboxyl terminus, similar to that of a rat prohormone, submandibular rat 1. We performed phylogenetic and sequence analysis of hCABS1. We found that from 72 CABS1 sequences currently annotated in the National Center for Biotechnology Information protein database, only 14 contain the anti-inflammatory domain “TxIFELL,” all of which are primates. We performed structural unfoldability analysis using PONDER and FoldIndex and discovered three domains that are highly disordered. Predictions of three-dimensional structure of hCABS1 using RaptorX, IonCom, and I-TASSER software agreed with these findings. Predicted neutrophil elastase cleavage density also correlated with hCABS1 regions of high structural disorder. Ligand binding prediction identified Ca²⁺, Mg²⁺, Zn²⁺, leucine, and thiamine pyrophosphate, a pattern observed in enzymes associated with energy metabolism and mitochondrial localization. These new observations on hCABS1 raise intriguing questions about the interconnection between the autonomic nervous system, stress, and the immune system. However, the precise molecular mechanisms involved in the complex biology of hCABS1 remain unclear. We provide a detailed in silico analysis of relevant aspects of the structure and function of hCABS1 and postulate extracellular and intracellular roles.     

Microscopic mechanism of DNA damage searching by hOGG1

The DNA backbone is often considered a track that allows long-range sliding of DNA repair enzymes in their search for rare damage sites in DNA. A proposed exemplar of DNA sliding is human 8-oxoguanine (^oG) DNA glycosylase 1 (hOGG1), which repairs mutagenic ^oG lesions in DNA. Here we use our high-resolution molecular clock method to show that macroscopic 1D DNA sliding of hOGG1 occurs by microscopic 2D and 3D steps that masquerade as sliding in resolution-limited single-molecule images. Strand sliding was limited to distances shorter than seven phosphate linkages because attaching a covalent chemical road block to a single DNA phosphate located between two closely spaced damage sites had little effect on transfers. The microscopic parameters describing the DNA search of hOGG1 were derived from numerical simulations constrained by the experimental data. These findings support a general mechanism where DNA glycosylases use highly dynamic multidimensional diffusion paths to scan DNA.     

Mouse Wnt9b transforming activity,tissue-specific expression,and evolution

The members of the Wnt family of secreted factors have oncogenic potential and important roles as developmental regulators. We report an analysis of mouse Wnt9b (also called Wnt15 and Wnt14b), including its cDNA sequence, chromosomal mapping, epithelial cell transforming activity, adult and embryonic tissue expression patterns, and evolution. We also deduced the full-length amino acid sequence of its close relative, Wnt9a (also called Wnt14), from unannotated genomic DNA sequences in GenBank. Full-length comparisons among Wnt amino acid sequences provide evidence that Wnt9b and Wnt9a are close paralogs of each other and are orthologs of Wnt9 genes from shark and hagfish. Mapping Wnt9b to The Jackson Laboratory BSS interspecific backcross panel places it at 63.0 cM on chromosome 11. Sequence comparisons of two pairs of linked Wnt genes (the Wnt9a-Wnt3a pair and the Wnt9b-Wnt3 pair) suggest that they arose from the relatively recent duplication of a single ancestral Wnt gene pair, confirming the close paralogous relationship of Wnt9a and Wnt9b. Wnt9b expression is primarily restricted to the kidney in the adult mouse, with lower levels detected in the preputial gland, liver, and mammary gland. Testing of staged whole mouse embryos from 9.5 to 17.5 days of gestation showed expression at all stages with a peak at day 10.5. In situ hybridization analysis showed expression in most but not all tissues of the 16.5-day embryo. No significant elevation of Wnt9b expression was detected in 29 mouse mammary tumor virus-induced tumors. Overexpression of Wnt9b in C57MG mammary epithelial cells caused small transformed foci in cell monolayers and a moderate morphological transformation in pooled colonies compared with Wnt1.     

Landscape scale genetic effects of habitat fragmentation on a high gene flow species: Speyeria idalia (Nymphalidae)

Detection of the genetic effects of recent habitat fragmentation in natural populations can be a difficult task, especially for high gene flow species. Previous analyses of mitochondrial DNA data from across the current range of Speyeria idalia indicated that the species exhibited high levels of gene flow among populations, with the exception of an isolated population in the eastern portion of its range. However, some populations are found on isolated habitat patches, which were recently separated from one another by large expanses of uninhabitable terrain, in the form of row crop agriculture. The goal of this study was to compare levels of genetic differentiation and diversity among populations found in relatively continuous habitat to populations in both recently and historically isolated habitat. Four microsatellite loci were used to genotype over 300 individuals from five populations in continuous habitat, five populations in recently fragmented habitat, and one historically isolated population. Results from the historically isolated population were concordant with previous analyses and suggest significant differentiation. Also, microsatellite data were consistent with the genetic effects of habitat fragmentation for the recently isolated populations, in the form of increased differentiation and decreased genetic diversity when compared to nonfragmented populations. These results suggest that given the appropriate control populations, microsatellite markers can be used to detect the effects of recent habitat fragmentation in natural populations, even at a large geographical scale in high gene flow species.     

A mutation in the pleckstrin homology (PH) domain of the FGD1 gene in an Italian family with faciogenital dysplasia (Aarskog-Scott syndrome)

Aarskog-Scott Syndrome (AAS) is an X-linked disorder characterised by short stature and multiple facial, limb and genital abnormalities. A gene, FGD1, altered in a patient with AAS phenotype, has been identified and found to encode a protein with homology to Rho/Rac guanine nucleotide exchange factors (Rho/Rac GEF). However, since this original report on identification of a mutated FGD1 gene in an AAS patient, no additional mutations in the FGD1 gene have been described. We analysed 13 independent patients with clinical diagnosis of AAS. One patient presented a mutation that results in a nucleotide change in exon 10 of the FGD1 gene (G2559>A) substituting a Gln for Arg in position 610. The mutation was found to segregate with the AAS phenotype in affected males and carrier females in the family of this patient. Interestingly, Arg-610 is located within one of the two pleckstrin homology (PH) domains of the FGD1 gene and it corresponds to a highly conserved residue which has been involved in InsP binding in PH domains of other proteins. The same residue is often mutated in the Bruton's tyrosine kinase (Btk) gene in patients with an X-linked agammaglobulinemia. The Arg610Gln mutation represents the first case of a mutation in the PH domain of the FGD1 gene and additional evidence that mutations in PH domains can be associated to human diseases.