Structure, organization and characterization of the gene cluster involved in the production of microcin E492, a channel-forming bacteriocin

Microcin E492 is a low-molecular-weight, channel-forming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post-translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492.     

High Prevalence of Human Cytomegalovirus Proteins and Nucleic Acids in Primary Breast Cancer and Metastatic Sentinel Lymph Nodes

Background

Breast cancer is a leading cause of death among women worldwide. Increasing evidence implies that human cytomegalovirus (HCMV) infection is associated with several malignancies. We aimed to examine whether HCMV is present in breast cancer and sentinel lymph node (SLN) metastases.

Materials and Methods

Formalin-fixed paraffin-embedded tissue specimens from breast cancer and paired sentinel lymph node (SLN) samples were obtained from patients with (n = 35) and without SLN metastasis (n = 38). HCMV immediate early (IE) and late (LA) proteins were detected using a sensitive immunohistochemistry (IHC) technique and HCMV DNA by real-time PCR.

Results

HCMV IE and LA proteins were abundantly expressed in 100% of breast cancer specimens. In SLN specimens, 94% of samples with metastases (n = 34) were positive for HCMV IE and LA proteins, mostly confined to neoplastic cells while some inflammatory cells were HCMV positive in 60% of lymph nodes without metastases (n = 35). The presence of HCMV DNA was confirmed in 12/12 (100%) of breast cancer and 10/11 (91%) SLN specimens from the metastatic group, but was not detected in 5/5 HCMV-negative, SLN-negative specimens. There was no statistically significant association between HCMV infection grades and progesterone receptor, estrogen receptor alpha and Elston grade status.

Conclusions

The role of HCMV in the pathogenesis of breast cancer is unclear. As HCMV proteins were mainly confined to neoplastic cells in primary breast cancer and SLN samples, our observations raise the question whether HCMV contributes to the tumorigenesis of breast cancer and its metastases.     

Silk scaffolds connected with different naturally occurring biomaterials for prostate cancer cell cultivation in 3D

In the present work, different biopolymer blend scaffolds based on the silk protein fibroin from Bombyx mori (BM) were prepared via freeze‐drying method. The chemical, structural, and mechanical properties of the three dimensional (3D) porous silk fibroin (SF) composite scaffolds of gelatin, collagen, and chitosan as well as SF from Antheraea pernyi (AP) and the recombinant spider silk protein spidroin (SSP1) have been systematically investigated, followed by cell culture experiments with epithelial prostate cancer cells (LNCaP) up to 14 days. Compared to the pure SF scaffold of BM, the blend scaffolds differ in porous morphology, elasticity, swelling behavior, and biochemical composition. The new composite scaffold with SSP1 showed an increased swelling degree and soft tissue like elastic properties. Whereas, in vitro cultivation of LNCaP cells demonstrated an increased growth behavior and spheroid formation within chitosan blended scaffolds based on its remarkable porosity, which supports nutrient supply matrix. Results of this study suggest that silk fibroin matrices are sufficient and certain SF composite scaffolds even improve 3D cell cultivation for prostate cancer research compared to matrices based on pure biomaterials or synthetic polymers.     

Are there links between responses of soil microbes and ecosystem functioning to elevated CO2, N deposition and warming? A global perspective

In recent years, there has been an increase in research to understand how global changes’ impacts on soil biota translate into altered ecosystem functioning. However, results vary between global change effects, soil taxa, and ecosystem processes studied, and a synthesis of relationships is lacking. Therefore, here we initiate such a synthesis to assess whether the effect size of global change drivers (elevated CO₂, N deposition, and warming) on soil microbial abundance is related with the effect size of these drivers on ecosystem functioning (plant biomass, soil C cycle, and soil N cycle) using meta‐analysis and structural equation modeling. For N deposition and warming, the global change effect size on soil microbes was positively associated with the global change effect size on ecosystem functioning, and these relationships were consistent across taxa and ecosystem processes. However, for elevated CO₂, such links were more taxon and ecosystem process specific. For example, fungal abundance responses to elevated CO₂ were positively correlated with those of plant biomass but negatively with those of the N cycle. Our results go beyond previous assessments of the sensitivity of soil microbes and ecosystem processes to global change, and demonstrate the existence of general links between the responses of soil microbial abundance and ecosystem functioning. Further we identify critical areas for future research, specifically altered precipitation, soil fauna, soil community composition, and litter decomposition, that are need to better quantify the ecosystem consequences of global change impacts on soil biodiversity.     

The outer membrane vesicles: Secretion system type zero

Gram‐negative bacteria have mechanisms through which they can colonize and survive in different environments, such as the secretion systems types (1‐6) that have been widely studied and characterized. Nowadays, some authors have proposed extracellular structures, such as the outer membrane vesicles (OMVs), to be considered as an additional and independent secretion system. The OMVs are spherical particles of 50‐250 nm in diameter; they originate in the outer membrane, and therefore they have a very similar composition to the latter. These particles can transport an important variety of biomolecules: enzymes, toxins, antigenic determinants and even nucleic acids. Thus, it is of great interest to collect data describing the advantages of the transport of biomolecules through the OMVs and, thus, determine their role as a potential secretion system.      

Retinal oxidative stress induced by high intraocular pressure

Glaucoma is an optic neuropathy in which retinal ganglion cells die probably through an apoptotic process. Apoptosis is known to involve free radicals in several systems including the retina. In this context, the aim of the present work was to analyze retinal oxidative damage in rats with glaucoma induced by the chronic injection of hyaluronic acid in the eye anterior chamber. The results showed a significant decrease in total retinal superoxide dismutase and catalase activities after 6 and 3 weeks of treatment with hyaluronic acid, respectively. Also, although GPX activity increased after 10 weeks of ocular hypertension, GSH levels significantly decreased at 6 weeks of treatment with hyaluronic acid. Moreover, retinal lipid peroxidation significantly increased in a time-of-hypertension-dependent manner. On the other hand, a significant decrease in both diurnal and nocturnal retinal melatonin content was detected at 3, 6, or 10 weeks of treatment with hyaluronic acid. The present results suggest that retinal oxidative stress may be involved in glaucomatous cell death. Thus, manipulation of intracellular redox status using antioxidants may be a new therapeutic tool to prevent glaucomatous neurodegeneration.     

Larger anti-adipogenic effect of angiotensin II on omental preadipose cells of obese humans

Objective: The ability to form new adipose cells is important to adipose tissue physiology; however, the mechanisms controlling the recruitment of adipocyte progenitors are poorly understood. A role for locally generated angiotensin II in this process is currently proposed. Given that visceral adipose tissue reportedly expresses higher levels of angiotensinogen compared with other depots and the strong association of augmented visceral fat mass with the adverse consequences of obesity, we studied the role of angiotensin II in regulating adipogenic differentiation in omental fat of obese and non‐obese humans. Research Methods and Procedures: The angiotensin II effect on adipose cell formation was evaluated in human omental adipocyte progenitor cells that were stimulated to adipogenic differentiation in vitro. The adipogenic response was measured by the activity of the differentiation marker glycerol‐3‐phosphate dehydrogenase. Results: Angiotensin II reduced the adipogenic response of adipocyte progenitor cells, and the extent of the decrease correlated directly with the subjects’ BMI (p = 0.01, R² = 0.30). A 56.3 ± 3.4% and 44.5 ± 2.7% reduction of adipogenesis was found in obese and non‐obese donors’ cells, respectively (p < 0.01). The effect of angiotensin II was reversed by type 1 angiotensin receptor antagonist losartan. Discussion: A greater anti‐adipogenic response to angiotensin II in omental adipose progenitor cells from obese subjects opens a venue to understand the deregulation of visceral fat tissue cellularity that has been associated with severe functional abnormalities of the obese condition.     

Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca²⁺-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca²⁺. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients.     

Extremely divergent histone H4 sequence from trypanosoma cruzi: Evolutionary implications

Trypanosoma cruzi presents six histones electrophoretically resolved in three gel systems. Indirect evidence shows that one of these histones, name, corresponds to H₄ in other species. We present evidence that histones is H₄ by sequencing its amino terminal end. The amino terminal of T. cruzi histone H₄, unlike that of other H₄s examined thus far is not blocked. Moreover, this protein presents two variants. This partial amino acid sequence of T. cruzi histone H₄ differs greatly from homologous sequences of human, yeast, or Tetrahymena. Since the conservatism of the core histones (H₂A, H₂B, H₃, and H₄) is clearly illustrated by comparative sequence analyses, the data shown here demonstrates that T. cruzi histone H₄ is the most divergent reported. Quantitative analysis of the data suggests that the rate of substitutions in the histone H₄ amino terminal sequence varies among different lineages. We postulate a slow-down in the evolutionary rate of histone H₄ amino terminal domain in the metazoa branch related perhaps to the appearance of a novel function for this domain.