Iodination of Xenopus laevis histone F2a1 in chromatin.

The reaction of calf thymus and Xenopus laevis histones with radioactive iodine has been studied under various conditions that affect chromatin structure. All histones from both species contain at least one tyrosine residue and, in a denaturing solvent, all the the histones react with iodine. Histone F2a1 has been studied in detail. Calf thymus F2a1 is known to contain four tyrosyls and all four react with iodine. In high voltage paper electrophoresis, the tyrosine-containing peptides from calf co-migrate with those from Xenopus F2a1, suggesting that the amino acid sequence is strongly conserved between these two species. Therefore, the published calf thymus F2a1 sequence has been used to order the Xenopus F2a1 peptides within the molecules. When gently isolated native chromatin is iodinated in a low ionic strength medium 60% of the radioactivity in F2a1 is in tyrosyl 88, 30% in tyrosyl 51, and tyrosyl 72 and 98 have almost no radioactivity. Reagents which remove the protein from the DNA (2 M NaCl) or partially disrupt protein tertiary structure (5 M urea) increase the reactivity of each of the four tyrosyls five- to tenfold, suggesting that all four are protected about equally by the overall folding of the chromatin. Isolated F2a1 iodinated in the presence of 10 M urea shows uniform labeling in each of the four peptides, suggesting that tyrosyl 72 and 98 are afforded some protection solely by protein-protein interactions. The stepwise removal of histones in increasing NaCl concentrations differentially increases the availability of each F2a1 tyrosyl. The preferential exposure of tyrosyl 88 coincides with the removal of the majority of F1 histones at 0.5 M NaCl while the gradual and stepwise increase in reactivity of tyrosyl 51, 72, and 98 correlates with the gradual removal of histones other than F1. Radioactive iodination of chromatin has been shown to be a sensitive probe for detecting changes in the association state (or conformation) of histone F2a1.     

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ , in the absence of molecular handles and using persistence length‐fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head‐to‐head orientation with a nonnative interface along their β‐strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.     

Biochemical characterization of beta2-adrenergic receptor dimers and oligomers

G Protein-coupled receptor dimerization/oligomerization has been well established during the last several years. Studies have demonstrated the existence of dimers/digomers both in vitro and in living cells. However, a thorough characterization of the biochemical nature of receptor dimers and oligomers as well as their occurrence at the cell surface has not been properly addressed. In this study, we show that both beta2-adrenergic receptor (beta2AR) dimers and oligomers exist at the plasma membrane and that the detection of such species, following receptor solubilization and resolution by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), does not result from the formation of spurious disulfide bonds during cell lysis. Moreover, our results indicate that the biochemical nature of beta2AR dimers is different from that of the oligomers. Although both complexes are partially resistant to SDS denaturation, disulfide bonding is absolutely required for the stability of beta2AR oligomers but not dimers in SDS-PAGE. Indeed, dimeric species can be detected even in the presence of high concentrations of reducing and alkylating agents. Although the different biochemical nature of the dimers and oligomers may be indicative of distinct biological roles in cells, additional studies will be required to further elucidate the biosynthesis and function of these receptor forms.     

The F1-F2 vowel chart for Czech whispered vowels a, e, i, o, u

Aim: The aim of this contribution is to present the formant chart of the Czech vowels a, e, i, o, u and show that this can be achieved by means of digital methods of sound processing. Method: A group of 35 Czech students of the Pedagogical Faculty of Palacky University was tested and a record of whispered vowels was taken from each of them. The record was digitalized and processed by the Discrete Fourier Trasform. The result is the power spectrum of the individual vocals - the graphic output consists of a plot of the relative power of individual frequencies in the original sound. The values of the first two maxima which represent the first and the second formants were determined from the graph. The values were plotted on a formant chart. Results: Altogether, 175 spectral analyses of individual vowels were performed. In the resulting power spectrum, the first and the second formant frequencies were identified. The first formant was plotted against the second one and pure vocal formant regions were identified. Conclusion: Frequency bands for the Czech vowel "a" were circumscribed between 850 and 1150 Hz for first formant (F1) and between 1200 and 2000 Hz for second formant (F2). Similarly, borders of frequency band for vowel "e" they were 700 and 950 Hz for F1 and 1700 and 3000 Hz for F2. For vowel "i" 300 and 450 Hz for F1 and 2000 and 3600 Hz for F2, for vowel "o" 600 and 800 Hz for F1 and 600 and 1400 Hz for F2, for vowel "u" 100 and 400 Hz for F1 and 400 and 1200 Hz for F2. Discussion: At low frequencies it is feasible to invoke the source-filter model of voice production and associate vowel identity with frequencies of the first two formants in the voice spectrum. On the other hand, subject to intonation, singing or other forms of exposed voice (such as emotional speech, focused speech), the formant regions tend to spread. In spectral analysis other frequencies dominate, so specific formant frequency bands are not easily recognizable. Although the resulting formant map is not much different from the formant map of Peterson, it carries basic information about specific Czech vowels. The results may be used in further research and in education.     

The relationship between histones F 2al and F 2a2 and the ancestral histone A peptide. Further evidence for the common origin of histones F 2al, F 2a2 and F 3.

The relationship between histones F 2al and F 2a2 becomes much more apparent if the alignment is not made between the total sequences but between the ancestral A peptide, reconstructed earlier for histone F 2al (IV) and F 2a2. 46.5% of the latter's sequence can thus be clearly connected with F 2al through this ancestral dodecapeptide. A parallel development of histones F 2al, F 2a2 and F 3 from the A peptide is proposed.     

Identification of subunits a, b, and c1 from Acetobacterium woodii Na+-F1F0-ATPase. Subunits c1, c2, AND c3 constitute a mixed c-oligomer

The Na(+)-F(1)F(0)-ATPase operon of Acetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunit a and b, a gene encoding a 16-kDa proteolipid (subunit c(1)), and two genes encoding 8-kDa proteolipids (subunits c(2) and c(3)). Because subunits a, b, and c(1) were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a, b, and c(1) are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunits a, c(2), c(3), b, delta, alpha, gamma, beta, and epsilon. Biochemical and immunological analyses revealed that subunits c(1), c(2), and c(3) are all part of the c-oligomer, the first of a F(1)F(0)-ATPase that contains 8- and 16-kDa proteolipids.     

The silent information regulator 3 protein, SIR3p, binds to chromatin fibers and assembles a hypercondensed chromatin architecture in the presence of salt

The telomeres and mating-type loci of budding yeast adopt a condensed, heterochromatin-like state through recruitment of the silent information regulator (SIR) proteins SIR2p, SIR3p, and SIR4p. In this study we characterize the chromatin binding determinants of recombinant SIR3p and identify how SIR3p mediates chromatin fiber condensation in vitro. Purified full-length SIR3p was incubated with naked DNA, nucleosome core particles, or defined nucleosomal arrays, and the resulting complexes were analyzed by electrophoretic shift assays, sedimentation velocity, and electron microscopy. SIR3p bound avidly to all three types of templates. SIR3p loading onto its nucleosomal sites in chromatin produced thickened condensed fibers that retained a beaded morphology. At higher SIR3p concentrations, individual nucleosomal arrays formed oligomeric suprastructures bridged by SIR3p oligomers. When condensed SIR3p-bound chromatin fibers were incubated in Mg(2+), they folded and oligomerized even further to produce hypercondensed higher-order chromatin structures. Collectively, these results define how SIR3p may function as a chromatin architectural protein and provide new insight into the interplay between endogenous and protein-mediated chromatin fiber condensation pathways.     

Mutual arrangement of histone H1 molecules in extended chromatin. Chymotryptic digestion of cross-linked H1 histone dimers

Mutual arrangement of histone H1 molecules in chromatin extended in low salt-EDTA buffer and additionally in the presence of urea was studied by means of reversible cross-linking combined with chymotryptic digestion. In the chromatins tested, the chymotryptic halves of H1 were cross-linked in all possible combinations; i.e., C-C, C-N and N-N. The results imply that the mutual arrangement of H1 histones is determined by the structure of extended nucleosomal chain, rather than chromatin superstructure.     

Implications of maternal nutrient restriction in transgenerational programming of hypertension and endothelial dysfunction across F1-F3 offspring

AimsAn extensive variety of prenatal insults are associated with an increased incidence of metabolic and cardiovascular disorders in adult life. We previously demonstrated that maternal global nutrient restriction during pregnancy leads to increased blood pressure and endothelial dysfunction in the adult offspring. This study aimed to assess whether prenatal exposure to nutritional insult has transgenerational effects in F₂ and F₃ offspring.Main methodsFor this, female Wistar rats were randomly divided into two groups on day 1 of pregnancy: a control group fed standard chow ad libitum and a restricted group fed 50% of the ad libitum intake throughout gestation. At delivery, all animals were fed a standard laboratory chow diet. At 11 weeks of age, one female and one male from each restricted litter were randomly selected and mated with rats from another restricted litters in order to generate the F₂ offspring. The same procedure produced F₃ generation. Similarly, the rats in the control group were bred for each generation.Key FindingsOur findings show that the deleterious effects of maternal nutrient restriction to which the F₀ mothers were exposed may not be limited to the male first generation. In fact, we found that elevated blood pressure, an impaired vasodilatory response to acetylcholine and alterations in NO production were all transferred to the subsequent males from F₂ and F₃ generations.SignificanceOur data show that global nutrient restriction during pregnancy results in a specific phenotype that can be passed transgenerationally to a second and third generation.     

Specificity of E2F1, E2F2, and E2F3 in mediating phenotypes induced by loss of Rb.

The Rb/E2F pathway plays a critical role in the control ofcellular proliferation. Here, we report that E2F1, E2F2, and E2F3 make major individual contributions toward the in vivo phenotypic consequences of Rb deficiency. In the developing lens of Rb(-/-) embryos, loss of E2F1, E2F2, or E2F3 reduces the unscheduled proliferation of fiber cells, with the loss of E2F3 having the most pronounced effect. In Rb-deficient retinas, all three E2Fs contribute equally to the ectopic proliferation of postmitotic neuronal cells. In contrast, E2F1 is unique in mediating apoptosis in both Rb(-/-) lenses and retinas. In the central nervous system, loss of E2F1 or E2F3 can almost completely eliminate the ectopic DNA replication and apoptosis observed in Rb(-/-) embryos, and loss of E2F2 partially reduces the unscheduled DNA replication and has no effect on apoptosis. These results provide clear evidence for functional specificity among E2Fs in the control of Rb-dependent proliferation and apoptosis in a tissue-specific manner.     

Investigation of DNA complexes with histones F3 and F3+F2a2]

Characteristic viscosity, sedimentation constant and optical anisotropy were studied of the complexes formed between DNA and histone fractions F3 and F3+F2a2. The parameters mentioned continuously change with the increase of protein content within the complex. Analysis of experimental data shows that binding of a histone bads to a decrease of size and thermodynamic rigidity of the DNA molecule. On the basis of results obtained a model of F3 histone binding with DNA is suggested, amino acid sequence of this protein being taken into account. Comparison of behaviour of nucleohistones DNA+F3 and DNA+F1 studied previously testifies different way of binding of these histones to DNA.     

The effect of the medium on the functional and structural properties of serum albumins. III. Relation between the N-F1-, F1-F2- and F2-E transitions of human serum albumin and temperature and ionic strength

Using fluorescence parameters of tryptophanyl and bound ANS, the acid-induced structural transitions of defatted monomeric human serum albumin were measured as pH-dependences from 6 to 2.5 in the wide range of temperature (10 to 45 degrees C) and ionic strength (from 0.001 to 0.2 M NaCl or 0.067 M Na2SO4). Temperature rise and decrease in ionic strength value result in the splitting of the N-F-transition onto two stages, N-F1 and F1-F2. The N-F1-transition is accompanied by the blue shift of tryptophanyl and ANS fluorescence spectra and increase in the ANS emission yield. The F1-F2-stage is manifested in an additional blue spectral shift and a sharp drop of the ANS emission yield, which is shown to be due to the lowering of albumin affinity for the dye. In the acidic-extension stage (F2-E), the spectra undergo a red shift which means that the nanosecond dipole relaxation of protein groups and bound water becomes faster. In the F2 from, the albumin affinity for ANS is significantly lowered; the association constant of the primary binding site is lower by an order of quantity and two secondary sites are practically disappeared. The complex effect of temperature, ionic strength and pH changes on the properties of ANS-binding sites is considered as a model of possible control influences of these factors upon the albumin transport of amphiphilic anions in organism.     

Regulation of the Pi-ATP exchange and hydrolytic reactions in F0-F1 reconstituted liposomes

The regulation of ATP hydrolysis and Pi-ATP exchange reactions by ATP, ADP, Mg2+, and phosphate was studied in liposomes containing F0-F1 obtained from bovine heart submitochondrial particles by solubilization with lauryl dimethylamino oxide as described previously (Dreyfus, G., Celis, H., and Ramirez, J. (1984) Anal. Biochem. 142, 215-220). A simultaneous analysis of ATP hydrolysis and the Pi-ATP exchange reactions showed that the ratio of hydrolysis/exchange is close to one when the ATP concentration is in the lower micromolar range. In this preparation ADP stimulates the Pi-ATP exchange reaction and depresses ATP hydrolysis. The exchange reaction is almost abolished when ADP is removed from the medium by an ATP-regenerating system. Mg2+ in millimolar concentrations stimulates Pi-ATP exchange, and at the same time decreases ATP hydrolysis; accordingly, the hydrolysis/exchange ratio depends on the concentration of Mg2+. Inorganic phosphate also controls this ratio, a lower ratio being observed at high phosphate concentrations. The Pi-ATP exchange reaction, but not ATP hydrolysis, depends on the concentration of medium phosphate. These results indicate that the kinetic characteristics of this F0-F1 preparation are modified by Mg2+, ATP, and phosphate.     

Glucosamine oligomers: 2. N.m.r. studies on a DP3.

This paper concerns the characterization of the chemical structure of a DP3 glucosamine oligomer. The assignments of nearly all protons are reported. Variations of 1H chemical shifts with pD and temperature are correlated to the pKa of amino groups, and not with substantial conformational changes.     

Influenza A Virus PB1-F2 Protein Expression Is Regulated in a Strain-Specific Manner by Sequences Located Downstream of the PB1-F2 Initiation Codon

Translation of influenza A virus PB1-F2 occurs in a second open reading frame (ORF) of the PB1 gene segment. PB1-F2 has been implicated in regulation of polymerase activity, immunopathology, susceptibility to secondary bacterial infection, and induction of apoptosis. Experimental evidence of PB1-F2 molecular function during infection has been collected primarily from human and avian viral isolates. As the 2009 H1N1 (H1N1pdm09) strain highlighted, some swine-derived influenza viruses have the capacity to infect human hosts and emerge as a pandemic. Understanding the impact that virulence factors from swine isolates have on both human and swine health could aid in early identification of viruses with pandemic potential. Studies examining PB1-F2 from swine isolates have focused primarily on H1N1pdm09, which does not encode PB1-F2 but was engineered to carry a full-length PB1-F2 ORF to assess the impact on viral replication and pathogenicity. However, experimental evidence of PB1-F2 protein expression from swine lineage viruses has not been demonstrated. Here, we reveal that during infection, PB1-F2 expression levels are substantially different in swine and human influenza viruses. We provide evidence that PB1-F2 expression is regulated at the translational level, with very low levels of PB1-F2 expression from swine lineage viruses relative to a human isolate PB1-F2. Translational regulation of PB1-F2 expression was partially mapped to two independent regions within the PB1 mRNA, located downstream of the PB1-F2 start site. Our data suggest that carrying a full-length PB1-F2 ORF may not be predictive of PB1-F2 expression in infected cells for all influenza A viruses.     

Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1

The influenza virus PB1-F2 is an 87-amino acid mitochondrial protein that previously has been shown to induce cell death, although the mechanism of apoptosis induction has remained unclear. In the process of characterizing its mechanism of action we found that the viral PB1-F2 protein sensitizes cells to apoptotic stimuli such as tumor necrosis factor alpha, as demonstrated by increased cleavage of caspase 3 substrates in PB1-F2-expressing cells. Moreover, treatment of purified mouse liver mitochondria with recombinant PB1-F2 protein resulted in cytochrome c release, loss of the mitochondrial membrane potential, and enhancement of tBid-induced mitochondrial permeabilization, suggesting a possible mechanism for the observed cellular sensitization to apoptosis. Using glutathione-S-transferase pulldowns with subsequent mass spectrometric analysis, we identified the mitochondrial interactors of the PB1-F2 protein and showed that the viral protein uniquely interacts with the inner mitochondrial membrane adenine nucleotide translocator 3 and the outer mitochondrial membrane voltage-dependent anion channel 1, both of which are implicated in the mitochondrial permeability transition during apoptosis. Consistent with this interaction, blockers of the permeability transition pore complex (PTPC) inhibited PB1-F2-induced mitochondrial permeabilization. Based on our findings, we propose a model whereby the proapoptotic PB1-F2 protein acts through the mitochondrial PTPC and may play a role in the down-regulation of the host immune response to infection.