SERPINA1 long transcripts produce non-secretory alpha1-antitrypsin isoform: In vitro translation in living cells.

SERPINA1 is a well-studied serpin gene due to its dramatic impact on human health. Translation initiation at the main SERPINA1 start codon produces the only known alpha1-antitrypsin (AAT) isoform intended for secretion. AAT performs essential functions by inhibiting proteases and modulating immunity. However, SERPINA1 expression at the level of translation is not sufficiently studied. Here we hypothesize that the main SERPINA1 ORF can be alternatively translated, producing a non-secretory AAT isoform by either masking or excluding a signal peptide. We defined SERPINA1 long mRNA isoforms specific for prostate (DU145) and liver (HepG2) cell lines and studied their individual expression by in vitro assay. We found that all long transcripts produce both glycosylated secretory AAT-eGFP fusion protein and non-glycosylated intracellular AAT-eGFP (initiated from an alternative AUG-2 start codon), with the proportion regulated by the SERPINA1 5'-UTR. Both fusion proteins localize to distinct cellular compartments: in contrast to a fusion with the secretory AAT accumulating in the ER, the intracellular one exhibits nuclear-cytoplasmic shuttling. We detected putative endogenous AAT isoform enriching the nuclear speckles. CONCLUSION: Alternative translation initiation might be a mechanism through which SERPINA1 expands the biological diversity of its protein products. Our findings open up new prospects for the study of SERPINA1 gene expression.

Soft X-ray Microscopy in Cell Biology: Current Status, Contributions and Prospects.

The recent advances achieved in microscopy technology have led to a significant breakthrough in biological research. Super-resolution fluorescent microscopy now allows us to visualize subcellular structures down to the pin-pointing of the single molecules in them, while modern electron microscopy has opened new possibilities in the study of protein complexes in their native, intracellular environment at near-atomic resolution. Nonetheless, both fluorescent and electron microscopy have remained beset by their principal shortcomings: the reliance on labeling procedures and severe sample volume limitations, respectively. Soft X-ray microscopy is a candidate method that can compensate for the shortcomings of both technologies by making possible observation of the entirety of the cellular interior without chemical fixation and labeling with an isotropic resolution of 40-70 nm. This will thus bridge the resolution gap between light and electron microscopy (although this gap is being narrowed, it still exists) and resolve the issue of compatibility with the former, and possibly in the near future, the latter methods. This review aims to assess the current state of soft X-ray microscopy and its impact on our understanding of the subcellular organization. It also attempts to look into the future of X-ray microscopy, particularly as relates to its seamless integration into the cell biology toolkit.

Towards unveiling the nature of short SERPINA1 transcripts: Avoiding the main ORF control to translate alpha1-antitrypsin C-terminal peptides.

Alternative ORFs in-frame with the known genes are challenging to reveal. Yet they may contribute significantly to proteome diversity. Here we focused on the individual expression of the SERPINA1 gene exon 5 leading to direct translation of alpha1-antitrypsin (AAT) C-terminal peptides. The discovery of alternative ways for their production may expand the current understanding of the serpin gene's functioning. We detected short transcripts expressed primarily in hepatocytes. We identified four variants of hepatocyte-specific SERPINA1 short transcripts and individually probed their potential to be translated in living cells. The long mRNA gave the full-length AAT-eGFP fusion, while in case of short transcripts we deduced four active SERPINA1 in-frame alternative ORFs encoding 10, 21, 153 and 169 amino acids AAT C-terminal oligo- and polypeptides. Unlike secretory AAT-eGFP fusion exhibiting classical AAT behavior, truncated AAT-fusions differ by intracellular retention and nuclear enrichment. Immunofluorescence on the endogenous AAT C-terminal epitope showed its accumulation in the cell nucleoli, indicating that short transcripts may be translated in vivo. FANTOM5 CAGE data on SERPINA1 suggest that short transcripts originate from the post-transcriptional cleavage of the spliced mRNA, initiated mainly from the hepatocyte-specific promoter. CONCLUSION: Short SERPINA1 transcripts may represent a source for the direct synthesis of AAT C-terminal peptides with properties uncommon to AAT.

Thymoquinone Induces Mitochondrial Damage and Death of Cerebellar Granule Neurons.

Thymoquinone (TQ) exhibits a wide spectrum of biological activities. Most studies on the neurotoxic action of TQ have been carried out in cancer cell lines. Here, we studied the toxic effect of TQ in primary neuronal cultures in vitro. Incubation with 0.04-0.05 mM TQ for 24 h induced the death of cultured cerebellar granule neurons (CGNs) in a dose-dependent manner. Neuronal death was preceded by an increase in the reactive oxygen species (ROS) generation, as demonstrated using CellROX Green and MitoSOX Red. Confocal and electron microscopy showed that incubation with 0.05 mM TQ for 5 h induced changes in the intracellular localization of mitochondria and mitochondria hypertrophy and cell swelling. The antioxidant N-acetyl-L-cysteine (2 mM) protected CGNs from the toxic action of TQ. Taken together, these facts suggest that TQ is toxic for normal neurons, while ROS-induced changes in the mitochondria can be one of the major causes of the TQ-induced neuronal damage and death.

Isolation of Large Amounts of Highly Pure Mitochondria for "Omics" Studies.

Ultracentrifugation on a density gradient remains the only reliable way to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method for isolation of mitochondria for proteomic studies that includes cell disruption by sonication, differential centrifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components, as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure in electron microphotographs.

TIP GROWTH OF NEUROSPORA CRASSA IN THE CONDITIONS OF THE RESOURCE DEFICIENCY: IMPAIRMENTS IN THE COHERENCE OF ELONGATION, BRANCHING, AND SEPTATION.

With the aid of intracellular fluorescent probes, peculiarities of elongation, branching, septation, and nucleus morphology have been studied in isolated 400-µm-long apical fragments of Neurospora crassa hyphae growing for several hours without the influx of the nutrient materials from the mycelium. We found that: 1) the diameter and growth rate of newly developing branches decreased as compared to intact mycelium (from 10-20 to 6.5 ± 0.4 µm and from 24 ± 1 to 6.7 ± 0.5 µm/min, respectively), but the initial orientation of growth did not change; 2) the branching rhythm was altered; abnormally long internode distances appeared (up to 1471 µm), and the development of some side-branch buds ceased; 3) the character of the septation remained unchanged, as well as the hyphal segment length (68 ± 2 µm). In hyphae growing for 4.5-5.0 h in isolation from the whole mycelium, the distance between the tip and the first septum was 210 ± 15 µm, which was comparable with the respective value in intact mycelium, whereas the mean distance between the growth tip and the first side branch (492 ±127 µm) and the dispersion of this parameter was considerably higher than in intact mycelium. In branches growing in isolation from the intact mycelium for 1.5-4.5 h, the length of the nucleus- free apical zones near the growth tip (from 5 to 33 µm) and the nucleus morphology were similar to the respective characteristics known for the N. crassa mycelium. The experimental model described here can be used for the investigations of molecular and genetic mechanisms regulating the interactions between intracellular structures involved in the tip growth of N. crassa.

[Lamellar Inclusion Bodies in the Retinal Pigment Epithelium of Diurnal Rodents].

The ultrastructure of the retinal pigment epithelium of a diurnal rodent (Brandt's vole) was described taking into account 1) the functions of the pigment epithelium as a participant in the renewal of photoreceptor outer segment and. 2) digestion of outer segment membranes into phagosomes of the retinal pigment epithelium. The myeloid bodies were observed after exposure of the pigment epithelium to light (200 lux, 4 hours) and darkness (0,1 lux, 1,5-hour). In the cytoplasm of the pigment epithelium of the vole no myeloid bodies were observed. Instead of it small lamellar bodies, which have the spiral form and size (from - 200 to 400 nm) were found. The structure of these lamellar bodies was described. Furthermore, the structures, which were presumably responsible for the transport of the digested material, were revealed. The evidence of it is the presence of 1) dense precipitate in the apical domain of the pigment epithelium and 2) microtubules which participate in transport of this precipitate.

Biogenesis of multilamellar membrane incorporations (myeloid bodies) in the Eolagurus luteus pigmented epithelium.

The study detected common ultrastructure of pigmented epithelium cells of Eolagurus luteus and other mammals and described its features characteristic of this species. Stages of interactions between the pigmented epithelium cells and external segments of retinal rod cells were studied. Migration of the external segments to the apical zone of a pigmented epithelium cell was observed. A pigmented epithelium cell was characterized by the presence of tubular endoplasmic reticulum and electron dense cytoplasm with processes directed to the retina. Multilamellar formations were detected, regarded as the initial stages of the myeloid body formation. These structures were characterized by periodicity of layers of ~4.2 and ~13.3 nm. The periodicity of layers in the formed myeloid body was ~23 nm. Interactions between the external segments and cells of pigmented epithelium, leading to formation of myeloid bodies, and the significance of this process for normal work of retinal elements are discussed.

[Organization of mitochondria in the growing hyphae of Neurospora crassa].

In vivo fluorescent labeling of mitochondria in Neurospora crassa showed the concentration of filamentous mitochondria within 30 µm of apex in growing hyphae. These mitochondrial assemblies propagated forward with the elongation of hyphae, split and segregated as the growing tip bifurcated and formed de novo when new branches formed farther away from the apex. The efficiency of the mitochondria concentration in the apical 30 µm zone is related to the growth rate and identical in hyphae cultivated in glucose- and sorbitol-containing media. The obtained data are discussed in connection with the behavior of microtubules in growing hyphae as well as with the electric heterogeneity of N. crassa hyphal apex described previously.

[A novel endogeneous inhibitor from hepatopancreas of Kamchatka crab (Paralithodes camtschaticus)].

A novel endogeneous inhibitor from hepatopancreas of Kamchatka crab (Paralithosed camtschaticus) was isolatyed. The inhibitor was purifeid through fractional affinity chromatography on gramicidin-diasorb followed by gel-filtration at Sephadex G-100. The inhibitor PC is a protein (M, 66 kDa) and active against serine collagenolytic protease PC at temperature optimum 15-20 degrees C, stable at 4-40 degrees C and was completely inactivated after heating to 50 degrees C and higher. 0.9-20% NaCl is necessary for its inhibitor activity. The inhibitor was found to slow down cell spreading in vitro in cell type-dependent manner. Fibroblasts are most prone to inhibitory effect, epithelial tumor derived cells show medium susceptibility, while fibrosarcoma cells were not affected.

[Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids].

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This "elementary" fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30-40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called "immature chromatin," which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.

Mitochondria as source of reactive oxygen species under oxidative stress. Study with novel mitochondria-targeted antioxidants--the "Skulachev-ion" derivatives.

Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1-2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of "bad" mitochondria in cell physiology is discussed.

[Chromatin structure within sites of DNA replication].

Conformational changes in chromatin structure are nowadays the object of intensive research due to its importance for proper regulation of intranuclear processes. The fine structure of chromatin within the DNA replication sites was studied in in situ fixed cells and cells permebilized by low ionic strength solutions in the presence of divalent cations. The latter method provides visualization of higher level chromatin structures such as globular chromomeres and chromonema fibres. Nascent DNA was detected immunochemically using anti-BrdU antibodies on the surface of ultrathin sections prepared from Epon-embedded material. It was shown that newly replicated DNA preferentially localized within the zones filled with globular and fibrillar elements with characteristic diameter of 30 nm, and not in chromonema fibres, while after replication had been completed DNA became embedded into as thick as 60-80 nm chromonema elements. The results obtained are discussed in the context of conception of hierarchical folding of chromatin fibers.

[Influence of membrane-active polyanions on various stages of the human cytomegalovirus life cycle in vitro].

Human cytomegalovirus (CMV), an agent of infection (CMVI), lethally dangerous for immune deficient neonates and adults was investigated in vitro as a target for a therapeutic effect of new membrane-active polyanionic compounds (MPC). Previous studies on the alicycle- and sulfate-modified carboxy-MPCs revealed a well-defined tendency of the anti-CMV activity amplification in parallel with increasing of the content of sulfate groups, enhancing the negative charge of the macromolecule. The dominating role of the electrostatic factor was confirmed by the highest activity of AS-688, compound with maximum sulfation among the tested MPCs. Its selectivity index (SI) of the CMVI inhibition in human diploid fibroblast cells reached 5450, 7500, 250 and 4286 in the microbicidal, viricidal, prophylactic and therapeutic schemes of the experiment respectively. The antiviral activity at the first, second and third schemes was explained by the polyanion-typical potential of electrostatic neutralization of the countercharged virions and prevention of the virus adsorption on the cell membranes (in competition with heparin sulfate, a cellular receptor of CMV), whereas the therapeutic effect required the ability of MPC to influence the intracellular stages of the CMV life cycle. The PCR and immunochemical assays revealed an inhibitory action of AS-688 on replication of the viral DNA and the following synthesis of the late viral protein gB with efficiency similar to that of gancyclovir (GCV). However, in contrast to GCV, acting as inhibitor of enzyme (viral RNA-polymerase) factor of the biosynthesis, the therapeutic activity of MPC could be interpreted by competition with viral RNA/DNA due to the specific character of the MPC molecular basis, initially constructed on the principle of nucleic acids backbone and charge adjustable imitation. This mechanism assuming reduction of the cytotoxicity risks, explained the experimentally observed fact of low cytotoxicity of MPCs and possible achievement of high SI. The MPC ability to penetrate into the cells without disruption of cellular membrane permeability was confirmed in experiments with the fluorescent-labeled derivate AS-679, structurally and functionally related to AS-688. In the light of the previously described HIV inhibiting properties of AS-688, AS-679 and MPC analogous, the results could be considered prospective in development of new highly effective agents for combined antiviral protection.

[The role of DNA methylation and histone modifications in structural maintainance of heterochromatin domains (chromocenters)].

The effects of DNA methylation inhibitor 5-azacytidine (5-aza-C) and histone acetylation inhibitor trichostatine A (TSA) on the structure of pericentric heterochromatin in cultured mouse cells (L929) has been studied. After 48 h of 5-aza-C treatment, about 85% of the cells demonstrate transformation of chromocenters from ovoid to elongated structures. Hypotonic treatment of these cells reveals tandemly arranged DAPI-positive globules, well distinguishable by light microscopy. The same globular units can be revealed in hypotonic-treated control cells. 48 h of TSA treatment causes dramatic decrease in HP 1alpha content in the cells. Chromocenters in 25% of treated cells became highly decondenced and can not be reliably detected by light and electron microscopy. 85% of cells demonstrate globular chromocenters with low HP 1alpha content. Hypotonic treatment causes transformation of compact chromocenters into ring-like structures, which can be either single or clustered. Rings are formed by uniform fiber, in which no globular subunits are detected. The data obtained are discussed concerning several mechanisms of heterochromatin structure maintenance and the roles of epigenetic marks in them.

Structural-functional model of the mitotic chromosome.

In the present review the structural role of noncoding DNA, mechanisms of differential staining of mitotic chromosomes, and structural organization of different levels of DNA compactization are discussed. A structural-functional model of the mitotic chromosome is proposed based on the principle of discreteness of structural levels of DNA compactization.