Age-related ultrastructural neurovascular changes in the female mouse cortex and hippocampus.

Blood-brain barrier (BBB) breakdown occurs in aging and neurodegenerative diseases. Although age-associated alterations have previously been described, most studies focused in male brains; hence, little is known about BBB breakdown in females. This study measured ultrastructural features in the aging female BBB using transmission electron microscopy and 3-dimensional reconstruction of cortical and hippocampal capillaries from 6- and 24-month-old female C57BL/6J mice. Aged cortical capillaries showed more changes than hippocampal capillaries. Specifically, the aged cortex showed thicker basement membrane, higher number and volume of endothelial pseudopods, decreased endothelial mitochondrial number, larger pericyte mitochondria, higher pericyte-endothelial cell contact, and increased tight junction tortuosity compared with young animals. Only increased basement membrane thickness and pericyte mitochondrial volume were observed in the aged hippocampus. Regional comparison revealed significant differences in endothelial pseudopods and tight junctions between the cortex and hippocampus of 24-month-old mice. Therefore, the aging female BBB shows region-specific ultrastructural alterations that may lead to oxidative stress and abnormal capillary blood flow and barrier stability, potentially contributing to cerebrovascular diseases, particularly in postmenopausal women.

Predominant synthesis of giant myofibrillar proteins in striated muscles of the long-tailed ground squirrel Urocitellus undulatus during interbout arousal.

Molecular mechanisms underlying muscle-mass retention during hibernation have been extensively discussed in recent years. This work tested the assumption that protein synthesis hyperactivation during interbout arousal of the long-tailed ground squirrel Urocitellus undulatus should be accompanied by increased calpain-1 activity in striated muscles. Calpain-1 is known to be autolysed and activated in parallel. Western blotting detected increased amounts of autolysed calpain-1 fragments in the heart (1.54-fold, p < 0.05) and m. longissimus dorsi (1.8-fold, p < 0.01) of ground squirrels during interbout arousal. The total protein synthesis rate determined by SUnSET declined 3.67-fold in the heart (p < 0.01) and 2.96-fold in m. longissimus dorsi (p < 0.01) during interbout arousal. The synthesis rates of calpain-1 substrates nebulin and titin in muscles did not differ during interbout arousal from those in active summer animals. A recovery of the volume of m. longissimus dorsi muscle fibres, a trend towards a heart-muscle mass increase and a restoration of the normal titin content (reduced in the muscles during hibernation) were observed. The results indicate that hyperactivation of calpain-1 in striated muscles of long-tailed ground squirrels during interbout arousal is accompanied by predominant synthesis of giant sarcomeric cytoskeleton proteins. These changes may contribute to muscle mass retention during hibernation.

Vitamin B12b Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells.

We have shown that hydroxycobalamin (vitamin В12b) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + В12b. It was found that cell death induced by DDC + B12b differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B12b used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.

Should the Treatment of Amyloidosis Be Personified? Molecular Mechanism of Amyloid Formation by Aß Peptide and Its Fragments.

Aß40 and Aß42 peptides are believed to be associated with Alzheimer's disease. Aggregates (plaques) of Aß fibrils are found in the brains of humans affected with this disease. The mechanism of formation of Aß fibrils has not been studied completely, which hinders the development of a correct strategy for therapeutic prevention of this neurodegenerative disorder. It has been found that the most toxic samples upon generation of fibrils are different oligomeric formations. Based on different research methods used for studying amyloidogenesis of Aß40 and Aß42 peptides and its amyloidogenic fragments, we have proposed a new mechanism of formation of amyloid fibrils. In accord with this mechanism, the main building unit for fibril generation is a ring-like oligomer. Association of ring-like oligomers results in the formation of fibrils of different morphologies. Our model implies that to prevent development of Alzheimer's disease a therapeutic intervention is required at the earliest stages of amyloidogenesis-at the stage of formation of ring-like oligomers. Therefore, the possibility of a personified approach for prevention not only of Alzheimer's disease development but also of other neurodegenerative diseases associated with the formation of fibrils is argued.

Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex.

Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers. Ultrastructural and super-resolution light microscopy showed that the CALHM1 channels were consistently associated with distinctive, large (1- to 2-µm) mitochondria spaced 20 to 40 nm from the presynaptic membrane. Pharmacological disruption of the mitochondrial respiratory chain limited the ability of taste cells to release ATP, suggesting that the immediate source of released ATP was the mitochondrion rather than a cytoplasmic pool of ATP. These large mitochondria may serve as both a reservoir of releasable ATP and the site of synthesis. The juxtaposition of the large mitochondria to areas of membrane displaying CALHM1 also defines a restricted compartment that limits the influx of Ca2+ upon opening of the nonselective CALHM1 channels. These findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles.

To Be Fibrils or To Be Nanofilms? Oligomers Are Building Blocks for Fibril and Nanofilm Formation of Fragments of Aß Peptide.

To identify the key stages in the amyloid fibril formation we studied the aggregation of amyloidogenic fragments of Aß peptide, Aß(16-25), Aß(31-40), and Aß(33-42), using the methods of electron microscopy, X-ray analysis, mass spectrometry, and structural modeling. We have found that fragments Aß(31-40) and Aß(33-42) form amyloid fibrils in the shape of bundles and ribbons, while fragment Aß(16-25) forms only nanofilms. We are the first who performed 2D reconstruction of amyloid fibrils by the Markham rotation technique on electron micrographs of negatively stained fragments of Aß peptide. Combined analysis of the data allows us to speculate that both the fibrils and the films are formed via association of ring-shaped oligomers with the external diameter of about 6 to 7 nm, the internal diameter of 2 to 3 nm, and the height of ∼3 nm. We conclude that such oligomers are the main building blocks in fibrils of any morphology. The interaction of ring oligomers with each other in different ways makes it possible to explain their polymorphism. The new mechanism of polymerization of amyloidogenic proteins and peptides, described here, could stimulate new approaches in the development of future therapeutics for the treatment of amyloid-related diseases.

An acid precipitation technique: A strip assay for the large-scale DNA polymerase activity screening.

A new format of a very rapid, low-cost and high-productive analysis based on the acid precipitation of radiolabeled DNA was developed. By contrast to the conventional processing of a large number of GF/C discs, the method employs one GF/C strip containing samples on individual teeth. The strip assay was validated by comparison with the glass fiber disk technique; the efficiency was demonstrated by screening E. coli superproducers and fractions obtained at the steps of Bst DNA polymerase, Large Fragment purification by the protocol we developed. The principle proposed allows simultaneous assaying many samples for the activity of different polymerases.

Isoform composition and gene expression of thick and thin filament proteins in striated muscles of mice after 30-day space flight.

Changes in isoform composition, gene expression of titin and nebulin, and isoform composition of myosin heavy chains as well as changes in titin phosphorylation level in skeletal (m. gastrocnemius, m. tibialis anterior, and m. psoas) and cardiac muscles of mice were studied after a 30-day-long space flight onboard the Russian spacecraft "BION-M" number 1. A muscle fibre-type shift from slow-to-fast and a decrease in the content of titin and nebulin in the skeletal muscles of animals from "Flight" group was found. Using Pro-Q Diamond staining, an ~3-fold increase in the phosphorylation level of titin in m. gastrocnemius of mice from the "Flight" group was detected. The content of titin and its phosphorylation level in the cardiac muscle of mice from "Flight" and "Control" groups did not differ; nevertheless an increase (2.2 times) in titin gene expression in the myocardium of flight animals was found. The observed changes are discussed in the context of their role in the contractile activity of striated muscles of mice under conditions of weightlessness.

Microorganisms form exocellular structures, trophosomes, to facilitate biodegradation of oil in aqueous media.

Cytochemical staining and microscopy were used to study the trophic structures and cellular morphotypes that are produced during the colonization of oil-water interfaces by oil-degrading yeasts and bacteria. Among the microorganisms studied here, the yeasts (Schwanniomyces occidentalis, Torulopsis candida, Candida tropicalis, Candida lipolytica, Candida maltosa, Candida paralipolytica) and two representative bacteria (Rhodococcus sp. and Pseudomonas putida) produced exocellular structures composed of biopolymers during growth on petroleum hydrocarbons. Four of the yeasts including S. occidentalis, T. candida, C. tropicalis and C. maltosa excreted polymers through modified sites in their cell wall ('canals'), whereas C. lipolytica and C. paralipolytica and the two bacterial species secreted polymers over the entire cell surface. These polymers took the form of fibrils and films that clogged pores and cavities on the surfaces of the oil droplets. A three-dimensional reconstruction of the cavities using serial thin sections showed that the exopolymer films isolated the ambient aqueous medium together with microbial cells and oil to form both closed and open granules that contained pools of oxidative enzymes utilized for the degradation of the oil hydrocarbons. The formation of such granules, or 'trophosomes,' appears to be a fundamental process that facilitates the efficient degradation of oil in aqueous media.