Heavy-Metal Stress and Developmental Patterns of Arbuscular Mycorrhizal Fungi

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species, Glomus etunicatum and Glomus intraradices, had different levels of sensitivity to metal stress. G. etunicatum was more sensitive to all three metals than was G. intraradices. A unique response of increased presymbiotic hyphal extension occurred in G. intraradices exposed to Cd and Pb. Presymbiotic hyphae of G. intraradices formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In G. intraradices grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by G. intraradices spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.     

Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans

Background

Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy.

Results

Initially, CGH was used to compare the approximately 100 kilobase MAT a and MATα mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MAT a) and the clinical isolate NIH12 (MATα) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains.

Conclusion

The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids.     

CK2 phosphorylation weakens 90 kDa MFP1 association to the nuclear matrix in Allium cepa

MFP1 is a conserved plant coiled-coil protein located on the stroma side of the chloroplast thylakoids, as well as in the nuclear matrix. It displays species-specific variability in the number of genes, proteins, and expression. Allium cepa has two nuclear proteins antigenically related to MFP1 with different M(r), pI, distribution, and expression, but only the 90 kDa MFP1 protein is a nuclear matrix component that associates with both the nucleoskeletal filaments and a new category of nuclear bodies. The 90 kDa AcMFP1 migrates in two-dimensional blots as two sets of spots. The hypo-phosphorylated forms (pI approximately 9.5) are tightly bound to the nuclear matrix, while high ionic strength buffers release the more acidic hyper-phosphorylated ones (pI approximately 8.5), suggesting that the protein is post-translationally modified, and that these modifications control its attachment to the nuclear matrix. Dephosphorylation by exogenous alkaline phosphatase and phosphorylation by exogenous CK2, as well as specific inhibition and stimulation of endogenous CK2 with heparin and spermine and spermidine, respectively, revealed that the protein is an in vitro and in vivo substrate of this enzyme, and that CK2 phosphorylation weakens the strength of its binding to the nuclear matrix. In synchronized cells, the nuclear 90 kDa AcMFP1 phosphorylation levels vary during the cell cycle with a moderate peak in G2. These results provide the first evidence for AcMFP1 in vivo phosphorylation, and open up further research on its nuclear functions.     

Stromal decidualization of endometriosis in the rhesus macaque (Macaca mulatta): a case report

During exploratory laparotomy, a 10-year-old female rhesus macaque was found to have a 6.0 x 9.5 x 2.0-cm multichambered, yellow, cystic mass cranial to the uterus, from which large amounts of opaque, white fluid were discharged into the abdominal cavity. The animal was euthanized, and the body was submitted for gross and histologic evaluation. Sections of the mass examined microscopically consisted of sheets of polygonal to round cells, with well defined cell borders and moderate amounts of eosinophilic cytoplasm. Scattered throughout these cells were few, variably sized glandular structures composed of columnar to cuboidal epithelium. Glandular epithelial cells were positive for keratin, and the sheets of polygonal cells were positive for vimentin and negative for keratin and CD 68. Gross and histologic appearance, immunohistochemical findings, and history of medroxyprogesterone acetate injections were compatible with a diagnosis of stromal decidualization of endometriosis. Subsequent biopsies of similar lesions in other rhesus macaques in the colony being treated with medroxyprogesterone acetate for endometriosis revealed comparable histologic findings.     

Decreased PLTP mass but elevated PLTP activity linked to insulin resistance in HTG: effects of bezafibrate therapy

Hypertriglyceridemia (HTG) is associated with insulin resistance, increased cholesteryl ester transfer (CET), and low HDL cholesterol. Phospholipid transfer protein (PLTP) may be involved in these relationships. Associations between CET, lipids, insulin resistance, CETP and PLTP activities, and PLTP mass were investigated in 18 HTG patients and 20 controls. Effects of 6 weeks of bezafibrate treatment were studied in HTG patients. HTG patients had higher serum triglycerides, insulin resistance, free fatty acid (FFA), and CET, lower levels of HDL cholesterol (-44%) and PLTP mass (-54%), and higher CETP (+20%) and PLTP activity (+48%) than controls. Bezafibrate reduced triglycerides, CET (-37%), insulin resistance (-53%), FFA (-48%), CETP activity (-12%), PLTP activity (-8%), and increased HDL cholesterol (+27%), whereas PLTP mass remained unchanged. Regression analysis showed a positive contribution of PLTP mass (P = 0.001) but not of PLTP activity to HDL cholesterol, whereas insulin resistance positively contributed to PLTP activity (P < 0.01). Bezafibrate-induced change in CET and HDL cholesterol correlated with changes in CETP activity and FFAs, but not with change in PLTP activity. Bezafibrate-induced change in PLTP activity correlated with change in FFAs (r = 0.455, P = 0.058). We propose that elevated PLTP activity in HTG is related to insulin resistance and not to increased PLTP mass. Bezafibrate-induced diminished insulin resistance is associated with a reduction of CET and PLTP activity.     

The Influence of Clinical Information in the Histopathologic Diagnosis of Melanocytic Skin Neoplasms

Background

We tested the relevance of clinical information in the histopathologic evaluation of melanocytic skin neoplasm (MSN).

Methods

Histopathologic specimens from 99 clinically atypical MSN were circulated among ten histopathologists; each case had clinical information available in a database with a five-step procedure (no information; age/sex/location; clinical diagnosis; clinical image; dermoscopic image); each step had a histopathologic diagnosis (D1 through D5); each diagnostic step had a level of diagnostic confidence (LDC) ranging from 1 (no diagnostic certainty) to 5 (absolute diagnostic certainty). The comparison of the LDC was employed with an analysis of variance (ANOVA) for repeated measures.

Findings

In D1 (no information), 36/99 cases (36.3%) had unanimous diagnosis; in D5 (full information available), 51/99 cases (51.5%) had unanimous diagnosis (p for difference between proportions <0.001). The observer agreement expressed as kappa increased significantly from D1 to D5. The mean LDC linearly increased for each observer from D1 through D5 (p for linear trend <0.001). On average, each histopathologist changed his initial diagnosis in 7 cases (range: 2–23). Most diagnostic changes were in D2 (age/sex/location).

Interpretation

The histopathologic criteria for the diagnosis of MSN can work as such, but the final histopathologic diagnosis is a clinically-aided interpretation. Clinical data sometimes reverse the initial histopathologic evaluation.     

The E3 Ubiquitin-Ligase Bmi1/Ring1A Controls the Proteasomal Degradation of Top2α Cleavage Complex – A Potentially New Drug Target

Background

The topoisomerases Top1, Top2α and Top2β are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2β are subject to proteasomal degradation, this phenomena was not demonstrated for Top2α.

Methodology/Principal Findings

We show here that Top2α is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2β. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2α degradation, increases the persistence of Top2α-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2α in-vitro and cellular overexpression of Bmi1 increases drug induced Top2α ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2α ubiquitination and drug-induced Top2α degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner.

Conclusions/Significance

The discovery that poisoned Top2α is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.     

Saturation of penicillin-binding protein 1 by β-lactam antibiotics in growing cells of Bacillus licheniformis

With the help of a new highly sensitive method allowing the quantification of free penicillin-binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β-lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.     

The role of the cytoskeleton in the formation of gap junctions by Connexin 30

Mutations in the genes that encode Connexin 26 (GJB2) and Connexin 30 (GJB6) are the most common known cause of hereditary nonsyndromic sensorineural deafness. Cx26 and Cx30 share a similar protein structure, as well as the same expression distribution pattern in the cochlea. Cx26 has different intracellular trafficking properties compared to those of Cx43 and Cx32, whose trafficking manner is consistent with the classical membrane protein secretory pathway. Until now, however, the trafficking patterns of Cx30 have not been studied. By means of an immunofluorescence staining approach, we found that the targeting of Cx30 to gap junctions in transfected HeLa cells is not affected by brefeldin A, suggesting a Golgi-independent feature, similar to Cx26. Nocodazole had a minimal effect on assembly and distribution of Cx30 gap junctions. Cytochalasin B-induced actin filament depolymerization, however, affected both the pattern and the distribution of Cx30 gap junctions. Co-localization with and/or interaction between Cx30 and microtubules and cortical actin filaments, but not with the tight/adherens junction protein ZO-1, was confirmed by immunofluorescence and/or immunoprecipitation methods. The results suggest that the cytoskeleton, and especially actin filaments, are important components in the processes of assembly, trafficking and stabilization of Cx30 gap junctions.     

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Background  

Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.