The difference in the lcrV sequences between Y. pestis and Y. pseudotuberculosis and its application for characterization of Y. pseudotuberculosis strains.

We have sequenced the lcrGVH operon from Y. pseudotuberculosis plasmid pYV995 and compared its sequence with that of Y. pestis. The sequences were highly homological, however, six base pair substitutions were found in one short 14 bp region termed variable sequence. Two oligonucleotides corresponding to variable sequence of Y. pestis (pes-V) or Y. pseudotuberculosis (ptb-V) were synthesized and were used as molecular probes in hybridization experiments with sets of Y. pestis and Y. pseudotuberculosis strains. All 17 Y. pestis strains tested were positive only with the pes-V probe, 18 of 21 Y. pseudotuberculosis strains were positive with the ptb-V probe, while three Y. pseudotuberculosis strains reacted with the pes-V probe but not the ptb-V probe. The 200 bp fragment including variable sequence was sequenced in seven Y. pseudotuberculosis strains. The Y. pseudotuberculosis strains which were positive with the pes-V probe possessed the 200 bp fragment sequence almost identical with that from Y. pestis. No correlation between the Y. pestis-like lcrV sequence and virulence was found for these strains. Moreover, the Y. pseudotuberculosis strains with Y. pestis-like sequences in contrast to Y. pestis possessed unaltered yadA gene. However, we have found the yadA frameshift mutation characteristic for Y. pestis in one Y. pseudotuberculosis strain 312.     

Method for three-dimensional visualization of neurodegeneration in cupric-silver stained serial rat brain slices

The spatial distribution of neurodegeneration in brains is difficult to visualize when working from 2-D serial slices. In studies where repetitive operant behavior measurements are made over several weeks following organic solvent exposure, definitive evidence of degeneration in brain structures may have been significantly cleared by the time the tissue is prepared histologically. The only remaining evidence that injury has occurred may be nothing more than neuronal and cellular debris. By choosing stains that are specific for this type of residual and/or indicative of specific pathology, a 3-D representation of the spatial distribution of the neuronal and cellular debris fields within the organ can be highlighted and displayed. We present a method for visualizing the spatial distribution of neuronal degeneration that can result from low-level organic solvent exposure scenarios. A cupric-silver stain highly specific for neuronal degeneration is used to identify neuronal debris fields in 73 serial slices of brains of rodents that were exposed to toluene vapors. Serial brain sections stained with cupric-silver are scanned at 600 dpi using a gray-scale protocol. Using commercially available software, scans are assembled into 3-D images showing both topographical and internal anatomical details. The reassembled images are further processed into stereo pairs. Gray-scale scans are compared to the original sections to establish gray-scale ranges for healthy and damaged tissue and artifact staining.     

Endogenous levels of serum estradiol and sex hormone binding globulin determine bone mineral density, bone remodeling, the rate of bone loss, and response to treatment with estrogen in elderly women

A total of 489 elderly women aged 65-75 yr who participated in a 3-yr, randomized, blinded osteoporosis trial underwent measurements of serum estradiol, bioavailable estradiol, and SHBG. At baseline, bone mineral density (BMD) was lower at the femoral sites (7-19%, P < 0.05), total body (6-8%, P < 0.05), and spine (5-9%, P = 0.2) in women in the lowest tertile for serum total estradiol [<9 pg/ml (33 pmol/liter)], serum bioavailable estradiol [<2.4 pg/ml (8.8 pmol/liter)], or highest tertile for serum SHBG (>165 nmol/liter), compared with women in the highest tertiles of total estradiol [>13.3 pg/ml (49 pmol/liter)] and bioavailable estradiol [>4 pg/ml (14 pmol/liter)] or lowest tertile for SHBG (<113 nmol/liter). Bone markers were increased in women in the lowest tertile for serum total estradiol (not significant) and bioavailable estradiol (P < 0.05) and highest tertile for SHBG (P < 0.05).In the longitudinal study, the rate of bone loss in the placebo group was significantly higher in total body (P < 0.05) and spine (P < 0.05) in women in the lowest tertile, compared with the highest tertile of serum bioavailable estradiol.After treatment with conjugated equine estrogens 0.625 mg/d, the increase in BMD was 4-6% higher at the femoral sites (P < 0.05), total body (P < 0.05), and spine (not significant), in the lowest tertile, compared with the highest tertile of serum bioavailable estradiol or highest tertile, compared with the lowest tertile of serum SHBG.In summary, small variations in endogenous serum estradiol and high serum SHBG determine differences in BMD and rate of bone loss in elderly women and also affect the response to treatment with estrogen. Women with a serum estradiol level of less than 9 pg/ml (33 pmol/liter) are optimal candidates for estrogen therapy for osteoporosis prevention.     

Docosahexanoic acid antagonizes TNF-α-induced necroptosis by attenuating oxidative stress,ceramide production,lysosomal dysfunction,and autophagic features

Objective

It was previously reported that docosahexanoic acid (DHA) reduces TNF-α-induced necrosis in L929 cells. However, the mechanisms underlying this reduction have not been investigated. The present study was designed to investigate cellular and biochemical mechanisms underlying the attenuation of TNF-α-induced necroptosis by DHA in L929 cells.

Methods

L929 cells were pre-treated with DHA prior to exposure to TNF-α, zVAD, or Necrostatin-1 (Nec-1). Cell death and survival were assessed by MTT and caspase activity assays, and microscopic visualization. Reactive oxygen species (ROS) were measured by flow cytometry. C16- and C18-ceramides were measured by mass spectrometry. Lysosomal membrane permeabilization (LMP) was evaluated by fluorescence microscopy and flow cytometry using Acridine Orange. Cathepsin L activation was evaluated by immunoblotting and fluorescence microscopy. Autophagy was assessed by immunoblotting of LC3-II and Beclin.

Results

Exposure of L929 cells to TNF-α alone for 24 h induced necroptosis, as evidenced by the inhibition of cell death by Nec-1, absence of caspase-3 activity and Lamin B cleavage, and morphological analysis. DHA attenuated multiple biochemical events associated with TNF-α-induced necroptosis, including ROS generation, ceramide production, lysosomal dysfunction, cathepsin L activation, and autophagic features. DHA also attenuated zVAD-induced necroptosis but did not attenuate the enhanced apoptosis and necrosis induced by the combination of TNF-α with Actinomycin D or zVAD, respectively, suggesting that its protective effects might be limited by the strength of the cell death insult induced by TNF-α.

Conclusions

DHA effectively attenuates TNF-α-induced necroptosis and autophagy, most likely via its ability to inhibit TNF-α-induced sphingolipid metabolism and oxidative stress. These results highlight the role of this Omega-3 fatty acid in antagonizing inflammatory cell death.     

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.     

An RNA virus hijacks an incognito function of a DNA repair enzyme

A previously described mammalian cell activity, called VPg unlinkase, specifically cleaves a unique protein-RNA covalent linkage generated during the viral genomic RNA replication steps of a picornavirus infection. For over three decades, the identity of this cellular activity and its normal role in the uninfected cell had remained elusive. Here we report the purification and identification of VPg unlinkase as the DNA repair enzyme, 5'-tyrosyl-DNA phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase activity in different mammalian cell lines correlates with their differential expression of TDP2. Furthermore, we show that recombinant TDP2 can cleave the protein-RNA linkage generated by different picornaviruses without impairing the integrity of viral RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host-pathogen interactions for this cellular enzyme. On the basis of the identification of TDP2 as a potential antiviral target, our findings may lead to the development of universal therapeutics to treat the millions of individuals afflicted annually with diseases caused by picornaviruses, including myocarditis, aseptic meningitis, encephalitis, hepatitis, and the common cold.     

Changes of the endothelium in cardiovascular pathology in the elderly. Part I. Signal molecules and endothelial functions

The review discusses the issue of the interaction between the endothelium and numerous biologically active substances, secreted by endotheliocytes and their environment--the so called signal molecules - in the most prevalent pathology in the elderly. In the elderly, signal molecules are synthesized by endothelial cells less intensively by comparison to young and middle-age people because a range of morphological features of endotheliocytes at elderly age: a hyperchromic nucleus; a smaller number of ribosomes, which leads to the inhibition of protein synthesis; a bigger number of lysosomes; structural changes in myofibrils; alterations in the mitochondrial structure such as crista death as well as the swelling and destruction of some organellas, which disturb the process of energosynthesis. At the same time, the main regulatory functions of the endothelium--vasomotor, antithrombocyte, anticoagulatory, thrombolytic, antiinflammatory, antioxidative, and antiproliferative ones--are the same in the healthy elderly and at earlier stages of ontogenesis. Disturbances in one or another endothelial function, especially at elderly age when the peculiarities of endothelial functioning under normal conditions are formed together with age-related morphological changes, result in fast development and progress of cardiovascular diseases.