Conversion of erythro-D-sphinganine to its [1-2H1] and [1-3H1] derivatives

A convenient chemical synthesis of erythro-D-[1-2H1] sphinganine and erythro-D-[1-3H1]sphinganine is described. The approach utilizes a stereospecific starting material (natural sphinganine prepared from bovine brain sphingomyelin) and applies a sequence of selective protection of functional groups yielding 2-acetamido-3-O-benzoyloctadecan-1-ol. Oxidation of the primary alcohol to an aldehyde followed by NaB2H4 or NaB3H4 reduction and hydrolysis of the protective groups yields erythro-D-[1-2H1]sphinganine or erythro-D-[1-3H1]sphinganine. The synthetic intermediates and isotopically labeled sphinganines are characterized by infrared analysis, 1H-nuclear magnetic resonance, optical rotation, and gas-liquid radiochromatographic and mass spectral fragmentation analyses. The [1-2H1] and [1-3H1] derivatives were obtained with overall yields (and isotope enrichments) of 11% (min. 84 mol% 2H1) and 8% (60 mCi/mmol), respectively.     

CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA

Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1–H3/H4 or H3/H4–DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)₂ tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.     

An Eight-Segment Swine Influenza Virus Harboring H1 and H3 Hemagglutinins Is Attenuated and Protective against H1N1 and H3N2 Subtypes in Pigs

Swine influenza virus (SIV) infections continue to cause production losses in the agricultural industry in addition to being a human public health concern. The primary method of controlling SIV is through vaccination. The killed SIV vaccines currently in use must be closely matched to the challenge virus, and their protective efficacy is limited. Live attenuated influenza vaccines (LAIV) provide strong, long-lived cell-mediated and humoral immunity against different influenza virus subtypes with no need for antigen matching. Here we report the generation of a new potential LAIV, an eight-segment SIV harboring two different SIV hemagglutinins (HAs), H1 and H3, in the genetic background of H1N1 SIV. This mutant SIV was generated by fusing the H3 HA ectodomain from A/Swine/Texas/4199-2/98 (H3N2) to the cytoplasmic tail, transmembrane domain, and stalk region of neuraminidase (NA) from A/Swine/Saskatchewan/18789/02 (H1N1) SIV. While this H1-H3 chimeric SIV, when propagated in vitro in the presence of exogenous neuraminidase, showed kinetics and growth properties similar to those of the parental wild-type virus, in vivo it was highly attenuated in pigs, demonstrating a great potential for serving as a dual LAIV. Furthermore, vaccination with the H1-H3 virus elicited robust immune responses, which conferred complete protection against infections with both H1 and H3 SIV subtypes in pigs.     

14N1H and 2H1H cross-relaxation in hydrated proteins.

The frequency dependence of the proton spin-lattice relaxation time T1 of solid hydrated bovine serum albumin and alpha-chymotrypsin has been measured over 4.5 decades in the range 10(4) to 3 X 10(8) Hz mainly by the aid of the field-cycling technique. The comparison between H2O- and D2O-hydrated samples permitted the distinction of exchangeable and unexchangeable protons. In all cases the 14N1H cross-relaxation dips due mainly to the amide groups have been observed. In addition, in the case of the deuterium exchanged proteins a 2H1H quadrupole dip appears. The amide groups act as relaxation sinks due to the coupling of the amide proton to 14N and adjacent protons. Outside of the dip regions the proton-proton coupling dominates. The fluctuations of the 14N1H and 1H1H interactions are of a different type. The unexchangeable protons show a T1 dispersion outside of the quadrupole dip regions given by the exceptional power law T1 approximately v0.75 +/- 0.05. It is shown that apart from structural information of the 14N spectra, 14N1H cross-relaxation spectroscopy permits the determination of correlation times in the range 10(-7) s less than tau less than 10(-4)S.     

Hexaploid H1 (ES) cells established from octaploid H1 cells are as DNA stable as pentaploid H1 cells

Hexaploid H1 (ES) cells (6H1 cells) were established from octaploid H1 cells (8H1 cells), as were pentaploid H1 cells (5H1 cells). 6H1 cells were compared with 5H1 cells. The number of chromosomes of 6H1 cells was 115, 20 more than the 95 of 5H1 cells. The durations of G(1), S, and G(2)/M phases of 6H1 cells were 3, 7, and 6 h, respectively, almost the same as those of 5H1 cells. The cell volume of 6H1 cells was equivalent that of 5H1 cells. The morphology of 6H1 cells was flattened circular cluster, different from the spherical cluster of 5H1 cells. 6H1 cells exhibited alkaline phosphatase activity as well as 5H1 cells. The DNA content of 6H1 cells was stable and maintained for 300 days of culturing, the same as that of 5H1 cells. The DNA stability of 6H1 cells was explained using a hypothesis concerning the DNA structure of polyploid cells because the asymmetric configuration of homologous chromosomes in 6H1 cells inhibited chromosome loss.     

Site-specificity of histone H1 methylation by two H1-specific protein-lysine N-methyltransferases from Euglena gracilis

1. The histone H1 fractions from rat spleen and liver were used as substrates for two H1-specific protein-lysine N-methyltransferases, V-A and V-B (protein methylase III) from Euglena gracilis. 2. When the enzymatically [methyl-3H]labeled H1 fractions were resolved by two-dimensional gel electrophoresis, four subtypes were found to be methylated (H1b, H1c, H1d and H1e). Both enzymes methylated H1c and H1b to approximately the same extent; H1d and H1e were methylated preferentially by enzyme V-B and V-A, respectively. 3. Histone H1c, [methyl-3H]labeled by the methyltransferase V-A, which had been digested by arginine-specific protease (Arg C protease), showed a single radioactive peptide on HPLC, indicating methylation site specificity of the enzyme. 4. Arg C protease-digestion of [methyl-3H]labeled H1c labeled by methyltransferase V-B indicated that this enzyme methylated two sites on the histone molecule. 5. The histone H1c methylation sites of these two enzymes did not overlap, indicating the two enzymes have different site specificity. 6. In combination with the other results, this suggests that the two enzymes serve discrete purposes, possibly involving the presumed different actions of histone H1 subtypes.     

Differences among chicken erythrocyte histones H1 and H5 in associating with H1-depleted polynucleosomes

1. Chicken erythrocyte histones H1a, H1b and H5 were associated to H1-depleted polynucleosomes from rat liver and the products were probed by digestion with micrococcal nuclease. 2. The digestion response of complexes with purified H1a or H1b resembled that of native polynucleosomes, while the digestion response of complexes with purified H5 exhibited specific different features--but none of these complexes resembled those with unfractionated histones H1a, H1b and H5. 3. However, after mixing purified histones H1a, H1b and H5 in the proportions (0.15:0.25:0.9) as these occur in erythrocyte nuclei and associating this mixture, the digestion response of the complexes was similar to that of the complexes with unfractionated histones.     

Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012

The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.     

H1 histones from mammalian testes : The widespread occurrence of H1t

H1t is a testis-specific H1 histone variant recently isolated from the rat [13]. We have now identified H1t in testicular extracts from mice, rabbits, hamsters, bulls, and boars, thus establishing its widespread presence in animals. H1t from all species could be differentiated from standard somatic H1 forms by a variety of criteria. It was poorly extracted by 5% trichloroacetic acid (TCA), migrated more rapidly than standard H1 forms during electrophoresis in the presence of sodium dodecyl sulfate (SDS), and eluted more slowly than standard H1 species during chromatography over cross-linked polyacrylamide beads (Bio-Gel P100). Using an improved purification procedure, H1t was isolated from two new species, mouse and rabbit. In these species as with the rat, H1t is readily identified by its low lysine (ca 19 mol%) and high arginine (6–7 mol%) content as compared with that of standard somatic H1 forms (ca 25% lysine and 3% arginine).     

Expression of histone 1 (H1) and testis-specific histone 1 (H1t) genes during stallion spermatogenesis

In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged expression up to elongating spermatids (Sd1) at stage V. A fragment of the equine H1t gene was cloned (GenBank Accession No. AJ865320). The mRNA expression of H1t was found at the level in spermatogonia and in primary spermatocytes up to mid-pachytene at stage VIII/I, whereas H1t protein was found to be expressed up to round spermatides (Sa/Sb1) at stage VIII/I. In peripubertal animals, the H1t protein expression was detected up to elongating spermatids (Sb2) at stage II. Analysis of testes of different ages (< or =2 years) and (> or =3 years) by real-time RT-PCR revealed an increase of H1t mRNA expression, with a wide range of individual variety between 2 and 4 years old animals indicating a stable expression in animals older than 4 years old. This is the first study to show the testis-specific H1t in the stallion and gives evidence that the well-known peripubertal infertility in the stallion may be related to an insufficient histone to protamine exchange. The pattern of protamine gene expression, however, has still to be elucidated.     

Complex patterns of human antisera reactivity to novel 2009 H1N1 and historical H1N1 influenza strains

Background

During the 2009 influenza pandemic, individuals over the age of 60 had the lowest incidence of infection with approximately 25% of these people having pre-existing, cross-reactive antibodies to novel 2009 H1N1 influenza isolates. It was proposed that older people had pre-existing antibodies induced by previous 1918-like virus infection(s) that cross-reacted to novel H1N1 strains.

Methodology/Principal Findings

Using antisera collected from a cohort of individuals collected before the second wave of novel H1N1 infections, only a minority of individuals with 1918 influenza specific antibodies also demonstrated hemagglutination-inhibition activity against the novel H1N1 influenza. In this study, we examined human antisera collected from individuals that ranged between the ages of 1 month and 90 years to determine the profile of seropositive influenza immunity to viruses representing H1N1 antigenic eras over the past 100 years. Even though HAI titers to novel 2009 H1N1 and the 1918 H1N1 influenza viruses were positively associated, the association was far from perfect, particularly for the older and younger age groups.

Conclusions/Significance

Therefore, there may be a complex set of immune responses that are retained in people infected with seasonal H1N1 that can contribute to the reduced rates of H1N1 influenza infection in older populations.     

Prophylactic and Therapeutic Efficacy of Avian Antibodies Against Influenza Virus H5N1 and H1N1 in Mice

Background

Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1.

Methods and Findings

We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection.

Conclusions

The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic.     

Separation of nucleosomes containing histones H1 and H5

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165–180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170–190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.     

Reduction of Hox gene expression by histone H1 depletion

The evolutionarily conserved homeotic (Hox) genes are organized in clusters and expressed collinearly to specify body patterning during embryonic development. Chromatin reorganization and decompaction are intimately connected with Hox gene activation. Linker histone H1 plays a key role in facilitating folding of higher order chromatin structure. Previous studies have shown that deletion of three somatic H1 subtypes together leads to embryonic lethality and that H1c/H1d/H1e triple knockout (TKO) embryonic stem cells (ESCs) display bulk chromatin decompaction. To investigate the potential role of H1 and higher order chromatin folding in the regulation of Hox gene expression, we systematically analyzed the expression of all 39 Hox genes in triple H1 null mouse embryos and ESCs by quantitative RT-PCR. Surprisingly, we find that H1 depletion causes significant reduction in the expression of a broad range of Hox genes in embryos and ESCs. To examine if any of the three H1 subtypes (H1c, H1d and H1e) is responsible for decreased expression of Hox gene in triple-H1 null ESCs, we derived and characterized H1c(-/-), H1d(-/-), and H1e(-/-) single-H1 null ESCs. We show that deletion of individual H1 subtypes results in down-regulation of specific Hox genes in ESCs. Finally we demonstrate that, in triple-H1- and single-H1-null ESCs, the levels of H3K4 trimethylation (H3K4me3) and H3K27 trimethylation (H3K27me3) were affected at specific Hox genes with decreased expression. Our data demonstrate that marked reduction in total H1 levels causes significant reduction in both expression and the level of active histone mark H3K4me3 at many Hox genes and that individual H1 subtypes may also contribute to the regulation of specific Hox gene expression. We suggest possible mechanisms for such an unexpected role of histone H1 in Hox gene regulation.     

Genetic polymorphism of human factor H (beta 1H)

Human Factor H (beta 1H) was found to be polymorphic after neuraminidase treatment and isoelectric focusing (IEF) under completely denaturing conditions. Three variants, FH 1, FH 2, and FH 3, were identified in a sample population of 81 unrelated caucasoid individuals. Family studies demonstrated correct mendelian segregation of FH 1, FH 2, and FH 3. Our data indicate that these genetic variants of human Factor H are encoded by three codominant alleles, FH*1, FH*2, and FH*3, at a single autosomal locus FH. In the sample analyzed, the gene frequencies of FH*1, FH*2, and FH*3 were, respectively, 0.691, 0.302, and 0.006.     

Genetic polymorphism of erythrocyte histone H1 in Japanese quail

Histone H1 from erythrocytes of Japanese quail was resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel into five fractions differing in apparent molecular weights. A polymorphism of histone H1.1, H1.2, and H1.3 bands was detected among quail individuals. While some birds possessed either a high (phenotype .3+) or a low (phenotype .3+/.3-) level of H1.3, at least half of the quail population lacked this H1 band (phenotype .3-). Appropriate genetic crosses demonstrated that H1.3 behaved as though it was coded by a gene with two codominant alleles at an autosomal locus. Using two-dimensional polyacrylamide gel electrophoresis (acid-urea followed by SDS gels), it was found that birds .3+ contained polypeptides H1.b1 and H1.b'1; birds .3-, polypeptides H1.b2 and H1.b'2 with lower apparent molecular weights; and birds .3+/.3-, both types of polypeptides in equal proportions. The H1.b2 + H1.b'2 complement was not discernible in SDS gels, for it migrated together with H1.c' within band H1.4. It was found that a small number of birds lacking the H1.2 band in SDS gels failed to express histone H1.a. Since birds with phenotype .2- with a defective allele of the gene H1.a were simultaneously lacking the H1.3 band, it seems that the imperfect allele of the H1.a gene might be closely linked to the alleles producing H1.b2 + H1.b'2.     

Isolation of the gene for the testis-specific H1 histone variant H1t

H1t is a testis-specific H1 variant found in pachytene spermatocytes and round spermatids of mammals. The H1t gene was isolated from the Sargent-Bonner library of recombinant lambda bacteriophage containing EcoRI fragments of rat liver DNA using a hybridization probe derived from a chicken H1 variant. The rat H1t gene encodes a 207-amino acid protein (ignoring the initiating methionine) that matches perfectly what is known of the sequence and composition of H1t isolated from rat testes. The gene lacks introns and has good matches to all the consensus sequences known to lie upstream from a variety of H1 genes from diverse organisms. It also has the standard downstream palindromic sequence that specifies the 3'-end of most histone messages. Accordingly, the features of the gene or its environs that restrict its expression to a particular phase of spermatogenesis are not yet evident.     

Testis-specific histone H1t is antigenically distinct among H1 subtypes

Histone H1t has been purified from rat testes and antibodies were elicited in rabbits. Immunoblotting studies with anti-histone H1t-IgG have shown that it reacted specifically with histone H1t but not with other histone H1 subtypes, namely H1a, -b, -c, -d, -e and H10. The anti-histone H1t-IgG also did not react with chicken erythrocyte histone H5. Immunoblotting studies have also revealed that the polyclonal anti-histone H1t-IgG reacted with (a) two polypeptide fragments, NBS-N and NBS-C, derived from N-bromosuccinimide cleavage of histone H1t, (b) two polypeptide fragments, CT-N and CT-C, derived from alpha-chymotrypsin cleavage of histone H1t, and (c) GH1t, globular domain of histone H1t obtained after trypsin cleavage. The indirect immunofluorescence studies on nuclei isolated from adult rat testes with anti-histone H1t-IgG showed that the fluorescence, particularly, of the pachytene nucleus was the brightest. On the other hand, anti-histone H1t-IgG did not stain nuclei from either liver or nuclei isolated from the testes of 10-day-old rats.