Binding of anti-nucleoside antibodies reveals different classes of DNA in the chromosomes of the kangaroo rat (Dipodomys ordii)

The binding of highly purified anti-nucleoside antibodies to fixed metaphase chromosomes of the kangaroo rat (Dipodomys ordii) revealed the presence of different classes of DNA in different regions of the chromosomes. To permit antibody binding, the chromosomal DNA was first made single-stranded by either ultraviolet irradiation, which denatures some classes of AT-rich DNA, or photo-oxidation, which denatures GC-rich DNA. The antibody binding patterns obtained matched the location of the different classes of satellite DNA in kangaroo rat chromosomes. After either denaturation method, anti-5-methylcytidine (anti-M) bound intensely only to the centromeric heterochromatic regions which are known to contain the GC rich, highly methylated HS-β satellite DNA of this species. The basic repeating unit of the HS-β sequence is 5′-ACACAGCGGG-3′ 3′-TGTGTCGCCC-5′ [4]. The binding of anti-M after UV irradiation is permitted by the production of pyrmidine (CC and TC) dimers in the right-hand portion of this repeating sequence, supporting the idea that the 5-methylcytosine residues are in this portion. After photo-oxidation, anti-cytidine (anti-C) and anti-adenosine (anti-A) also showed intense binding to the centromeric heterochromatin. In addition, these antibodies showed moderately intense binding to non-centromeric heterochromatic regions, which contain the relatively GC-rich HS-α and MS satellite DNAs. After UV irradiation, anti-A binding produced a banding pattern identical to the quinacrine (Q-band) pattern, with bright chromosome arms and very dull centromeric heterochromatic regions, while anti-C showed moderate binding in the centromeric regions and fairly even but weak binding elsewhere.The results have clarified the way in which anti-nucleoside antibodies react with chromosomal DNA. The reactivity of anti-A, anti-C and anti-M with the partially denatured HS-β satellite DNA supports the idea that antibody binding requires denaturation of a sequence perhaps no more than 5 base pairs long. In addition, it appears that it is not necessary to have more than one identical base in a row to permit antibody binding.     

Research

Gram-positive bacterium Streptococcus gordonii, a human oral commensal, was engineered to display a single-chain Fv (scFv) antibody fragment at the cell surface. The previously developed host-vector system allowed expression of the Guy’s 13 scFv as a fusion with the streptococcal surface protein M6. Surface expression of the 515-amino acid M6/scFv fusion protein was confirmed by Western blot analysis on cellular fractions and flow cytometric analysis. Guy’s 13 scFv was derived from the Guy’s 13 monoclonal antibody, which was raised against streptococcal antigen I/II (SA I/II), the major adhesin of the caries-producing bacterium Streptococcus mutans. Surface plasmon resonance was used to test binding of scFv-expressing S. gordonii to SA I/II. Whole cells of recombinant S. gordonii were found to specifically bind to immobilised SA I/II and binding was inhibited by fluid-phase SA I/II in a dose-dependent manner. Production of a functional scFv in S. gordonii is the first step towards the development of genetically engineered commensal bacteria that, by colonizing mucosal surfaces, may provide the host with sustained delivery of recombinant antibodies.     

RE-EXAMINATION OF FERTILIZATION INHIBITION STUDIES WITH ANTI-FROG-EGG-JELLY ANTIBODIES: EXPERIMENTS WITH NON-PRECIPITATING ANTIBODIES

Antisera were prepared in rabbits against oviducal and egg-jellies of the frog, Rana japonica . Gamma-globulin fractions from the antisera were degraded to a univalent, non-precipitating form by a papain digestion-reduction procedure. Agar diffusion analyses proved that the digested antibody fragments were inhibitory to the precipitin reaction of undigested, multivalent antibodies with jelly antigens. The treatment of unfertilized eggs with undigested, multivalent antibodies resulted in a significant loss of egg-fertilizability. In contrast, treatment with the univalent fragments of antibodies did not affect egg-fertilizability, similarly to the treatment with both univalent and multivalent γ-globulins from control, non-immune sera. Fertilization was inhibited in large measure when unfertilized eggs were subjected to a dual treatment with univalent antibodies and γ-globulins from anti-rabbit γ-globulin sheep serum. The inhibition of fertilization in the above experiments was always accompanied by the formation of a precipitation layer at the surface of the jelly envelopes. It is concluded that the failure of fertilization in the multivalent antibody-treated eggs results from a secondary effect rather than a specific blocking of a sperm-jelly interaction essential for fertilization.     

Assessment of Human Immune Response to Mutans Streptococcal Glucosyltransferase Peptides Selected by MHC Class II Binding Probability

Dental decay is a major public health challenge, causing substantial social and economic burdens. In animals, vaccination against mutans streptococci, the causative organism, interferes with dental caries. The mutans streptococcal glucosyltransferase (GTF) has been effectively used as a protein antigen in experimental dental caries vaccines. Compared to whole proteins, peptide subunits can focus immune responses on protective epitopes, and not on potentially harmful cross reactive antigens. In the past we selected peptide subunits of GTF for vaccine discovery based on putative functional significance and conservation of GTF primary structure. To focus on the immunogenicity of peptides, we estimated the probability of MHC class II binding. Twenty 20-mer linear GTF peptides were synthesized on this basis and their immunoreactivity explored. Significant human peripheral blood mononuclear cell (PBMC; n = 12) proliferation was observed in response to amino acids (AA) 502–521 (peptide 7), located in the catalytic domain of GTF. Human serum (n = 36) antibody reactivity was observed to AA 438–457 (peptide 5), AA 502–521 (peptide 7), and AA 1376–1395 (peptide 16). Whole saliva mutans streptococcal levels were used as markers of mutans infection, and dental examinations to determine existing and historic caries (DMFS score) were performed. DMFS scores correlated with mutans streptococcal counts, but not with immune responses. We have identified peptides with projected avid MHC-binding activity that reacted with human PBMC and serum antibody, implying that these peptides are immunogenic and may be of significance in a subunit dental caries vaccine.     

Neutralizing Activities of Early and Late IgG Fragments from Rabbits Immunized with Herpes Simplex Virus

Rabbits immunized with herpes virus were bled periodically and bivalent and univalent fragments of IgG from each serum sample were prepared by enzymatic digestion. The 2-week F(ab′)₂ showed a low neutralizing activity only after addition of anti-IgG. F(ab′)₂ of the 4-week serum retained almost all of the neutralizing activity of IgG, while its univalent fragments demonstrated none even when tested with anti-IgG. In contrast to these early IgG fragments, univalent fragments of the 9-week and 20-week IgG neutralized the virus to considerable extents in the absence of anti-IgG; after addition of anti-IgG the activity equaled that of intact IgG in the cases of Fab′ and Fab-II, though the activity of Fab-I was relatively low. The three univalent fragments were all sensitive to heating at 70 C and to ultraviolet irradiation, whereas intact IgG resisted these treatments. F(ab′)₂ was resistant to the heating and less sensitive to ultraviolet irradiation than univalent fragments. Neutralization kinetic curve experiments to test blocking effects of IgG fragments against the neutralization by intact IgG suggested that the early Fab′ did combine with the virus and that the late Fab′ exerted a higher blocking effect than the early Fab′.     

Improvement of the Antitumor Activity of Black Currant Polysaccharide by an Enzymatic Treatment

A polysaccharide-rich substance isolated from black currant, named cassis polysaccharide (CAPS), was partially digested with β-galactosidase from Aspergillus oryzae and its immunostimulatory activity was investigated. The in vitro cytokine-inducing effect of CAPS on RAW264 cells was gradually decreased along with lowering of the average MW of CAPS. In vivo, partially digested CAPS with a mean MW of approximately 20,000 showed the most potent antitumor activity against Ehrlich carcinoma in mice.     

Mutanase from <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. MP-1 produced inductively by fungal α-1,3-glucan and its potential for the degradation of mutan and <Emphasis Type="Italic">Streptococcus mutans</Emphasis> biofilm

Laetiporus sulphureus is a source of α-1,3-glucan that can substitute for the commercially-unavailable streptococcal mutan used to induce microbial mutanases. The water-insoluble fraction of its fruiting bodies from 0.15 to 0.2% (w/v) induced mutanase activity in Paenibacillus sp. MP-1 at 0.35 μ ml⁻¹. The mutanase extensively hydrolyzed streptococcal mutan, giving 23% of saccharification, and 83% of solubilization of glucan after 6 h. It also degraded α-1,3-polymers of biofilms, formed in vitro by Streptococcus mutans, even after only 3 min of contact.     

Prokaryotic expression and characterization of avian influenza A virus <Emphasis Type="Italic">M2</Emphasis> gene as a candidate for universal recombinant vaccine against influenza A subtypes; specially H5N1 and H9N2

The conserved M2 protein of influenza A virus is considered as a promising candidate target for a broad-spectrum, recombinant influenza A vaccine. In the present study, the open reading frame (ORF) of avian influenza A/chicken/Iran/101/1998 (H9N2) M2 gene was amplified then cloned in pAED4, prokaryotic expression vector. M2 protein was produced through the expression of this recombinant expression vector (pAED4-M2) in E. coli BL21 (DE3) strain. The expressed M2 protein was analyzed on SDS-PAGE. Western blot assay was used to examine the immunoreaction of the expressed protein using commercial polyclonal anti-M2 antibody. The antigenicity and biological activity of the recombinant protein was also qualitatively detected on infected MDCK cells surface by immunofluorescence assay using rabbit’s immunized antiserum. So, according to the sequence alignment based on the mentioned isolate and the result of immunoassay reaction, it seems recombinant vaccine based on A/chicken/Iran/101/1998(H9N2) M2 protein isolate might cover majority of influenza A virus strains specially H5 and H9 circulating in Iran and neighbor regions significantly.     

On the application of polyelectrolyte "limiting laws" to the helix-coil transition of DNA. II. The effect of Mg++ counterions

The techniques of the previous article are here applied to the case for which the solution contains, in addition to excess uni–univalent salt, one equivalent of divalent counterions per mole nucleotide. In agreement with the melting temperature measurements of Dove and Davidson for Mg⁺⁺, it is predicted that a region of uni–univalent salt concentration then exists in which (dT _m/^d log m A +) is negative. It is further predicted, in accord with experiment, that in the presence of divalent counterions, the helical form of DNA is much more stable than in their absence.     

Aggregated Form of Dextransucrases from Leuconostoc mesenteroides NRRL B-512F and Its Constitutive Mutant

Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively, DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS–PAGE were all 170kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V₀) on Sepharose 6B, while the DSM-G was eluted at 1.2 × V₀ volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.     

The eects of a ring chromosome on the meiotic segregation of other chromosomes in Saccharomyces cerevisiae

Meiotic chromosome segregation must occur with high fidelity in order to prevent the generation of aneuploid cells. We have previously described the identification and genetic characterization of a yeast mutant with defects in meiotic sister-chromatid segregation. We attributed the phenotype in this mutant to a dominant allele, which we referred to as SID1-1. These mutants appeared to exhibit high levels of nondisjunction and precocious separation of sister-chromatids of chromosome III, as well as precocious separation of sister chromatids of chromosome VIII and a univalent artificial chromosome. We show here that the unusual meiotic behavior of chromosome III in these strains is due to the presence of a ring III chromosome, rather than a mutant gene. Additional experiments demonstrate that a ring III/rod III pair alters the meiotic segregation of a univalent artificial chromosome.     

Therapeutic effect of llama derived VHH fragments against Streptococcus mutans on the development of dental caries

Streptococcus mutans is the main cause of dental caries. We evaluated the therapeutic effect of variable regions of a llama heavy chain antibody fragments directed against S. mutans named S36-VHH (S for Streptococcus) alone or fused with glucose oxidase (GOx) from Aspergillus niger. Western blot analysis and ELISA revealed binding of the S36-VHH to the streptococcal antigen I/II adhesin molecule of S. mutans serotype C. In a rat-desalivated caries model, daily administration of S36-VHH significantly reduced the development of smooth surface caries. No additional therapeutic effect of GOx was observed. Our results suggest that llama VHH antibodies may be a potential benefit as prophylaxis against dental caries.     

Direction of transcription affects the replication mode of lambda in an in vitro system

Summary pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably mantained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30° C. The optimal concentration of template DNA was 40 g/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its serivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs replicated in the E. coli in vitro system were monomeric supercoiled DNA forms that had completed at least one round of replication, although a set of putative replicative intermediates could also be found. The results suggest that a specific plasmid-encoded factor is needed for the replication of the streptococcal plasmids.     

Effectivein vivo induction of lymphokine-activated killer (LAK) cells by pretreatment with a streptococcal preparation,OK-432

Effects of a streptococcal preparation, OK-432, on precursors of lymphokine-activated killer (LAK) cells were observedin vivo. Total number of splenocytes and the ratio of asGM ₁ ⁺ cells increased gradually after i.v. administration of OK-432, reaching their peaks at 3 to 4 days. It was found that as GM ₁ ⁺ cells were nonadherent and large in size. There were little differences in the ratios of Thy-1⁺, Lyt-2⁺, and L3T4⁺ cells before and after OK-432 treatment. Mice were injected i.p. with recombinant interleukin 2 (rIL-2) at a dose of 5 × 10⁴ U per mouse 4 days after OK-432 administration and LAK activity in their splenocytes was examined using natural killer (NK) resistant EL-4 target cells. Splenocytes in mice treated with both OK-432 and rIL-2 showed higher LAK activity than those in mice treated with rIL-2 alone.In vivo treatment with anti asGM, antibody prior to rIL-2 injection abolished completely such augmentation of LAK activity in OK-432 treated mice. These results demonstrated that asGM ₁ ⁺ LAK precursor cells induced by OK-432 were effectively differentiated into LAK cells by rIL-2.     

Challenges with afucosylation content in antibody‐based drugs: Guidance provided by mathematical modeling

The theory of competitive ligand–receptor binding has been used to analyze the effect of afucosylation‐based antibody heterogeneity on Fc‐FcγRIIIa ligand–receptor binding activity. In vitro activity is found to represent a linear combination of the component antibody activities, weighted by the relative concentrations of the different afucosylated antibody forms. An analysis of ELISA binding activity data has allowed for the dissection of the activity contributions of the different afucosylated antibodies, revealing that the heterogeneous afucosylated antibody exhibits greater activity, on a per mole basis, when compared to the homogeneous afucosylated antibody. The ratio of the afucosylated antibody equilibrium dissociation constants is computed to be K_AF/K_A ≈ 0.6–0.9, where K_AF and K_A denote the dissociation equilibrium constant of the heterogeneous and the homogeneous afucosylated antibodies, respectively. Our analysis also reveals that, in general, activity scales quadratically with the afucosylated glycan content of a sample. Linear activity–afucosylated glycan fraction correlations reported in the literature are shown to represent specific cases of this general scaling and result from oversimplifying the underlying antibody concentration distributions. The implications of our findings for drug development are also discussed. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:775–782, 2015     

Isolation and partial characterization of a type II Fe receptor from a group A streptococcus

A group A streptococcal strain rich in Fc receptors was selected by an immunoblotting technique and used as the source for isolation of a functionally active Fc receptor. A variety of extraction techniques were compared including (1) heat extraction at neutral, acid or alkaline pH, (2) treatment with the enzymes mutanolysin, hyaluronidase, trypsin, papain or phage lysin, or (3) autoclaving or heating in the presence of sodium dodecyl sulfate. The most homogeneous receptor was recovered following heat extraction and contained two molecular weight forms. The major form had a molecular weight of 56 000 daltons and the minor form had a molecular weight of 38 000 daltons. These two proteins could be isolated without loss of activity by binding to and elution from a column of immobilized human IgG. An antibody prepared against a single form of the affinity purified receptor demonstrated reactivity with both molecular weight forms of the heat extracted receptor. The group A receptor was found to be both antigenically and physicochemically distinct from either the type I receptor found on the majority of Staphylococcus aureus strains or the type III Fc receptors found on the majority of group C streptococcal strains.     

In vitro compared activity of telithromycin and azithromycin against northwest Italian isolates of Streptococcus pyogenes and Streptococcus pneumoniae with different erythromycin susceptibility

Aims: This study compared the in vitro activity of telithromycin with that of azithromycin against 438 Streptococcus pyogenes and 198 Streptococcus pneumoniae, isolated over the period 2005–2007 from specimens of different human origin obtained in three Piemonte Region’s hospitals. Methods and Results: The determination of antimicrobial activity was evaluated by the microdilution broth method and the erythromycin‐resistant (Ery‐R) phenotypes by the triple‐disc test. Exactly 78·8% of S. pyogenes and 69·2% of S. pneumoniae were erythromycin‐susceptible (Ery‐S). Concerning S. pyogenes, telithromycin was active against M and inducible MLS_B, subtype‐C, phenotypes but not against constitutive MLS_B strains. Telithromycin acted well against all S. pneumoniae, irrespective of their mechanism of macrolide‐resistance. On the contrary, the Ery‐R isolates, both S. pyogenes and S. pneumoniae, were resistant to azithromycin. Conclusions: Our results indicate that macrolide resistance in streptococci still persist in northwest Italy (21·2% of S. pyogenes and 30·8% of S. pneumoniae) and that telithromycin is confirmed as being extremely active even against recent clinical Ery‐R streptococcal isolates. Significance and Impact of the Study: The present study emphasizes that an active surveillance of the phenotype distribution and antibacterial resistance in streptococci is essential in guiding the effective use of empirical treatment option for streptococcal infections, also at regional level.     

Reticulate evolution of the hybrid produced artificially by crosses between Osmunda banksiifolia and Osmunda lancea

Although ferns have been developed by hybridization and chromosome doubling, no natural polyploidy has yet been recorded in Osmundaceae. So, we produced hybrids artificially by crosses between Osmunda banksiifolia (2n = 2x = 44) and Osmunda lancea (2n = 2x = 44), and investigated their sporogenesis. From the O. banksiifolia × O. lancea hybrid with 44 univalent chromosomes, allotetraploids with 44 bivalent chromosomes were produced by chromosome doubling, and allotriploids with 22 univalent chromosomes and 22 bivalent chromosomes were then produced by back crosses. The results show when and how chromosome doubling occurs in hybrids. The success of artificial hybridization between O. banksiifolia and O. lancea, did not, however, reflect any product of natural hybridization between the two species.     

Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE‐B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two‐component system, which negatively regulates many virulence factor genes, resulting in a hyper‐virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression.     

Meiosis of T70H translocation trisomic male mice

Meiosis of T70H/+, Ts(1¹³)70H translocation trisomic male mice has been studied using C-banded preparations and ³H-thymidine autoradiography of the first meiotic division. Epididymal sperm counts and sperm morphology scores were also collected. As reported earlier, at the first meiotic division the translocation involved chromosomes 1, 13, 13¹ and 1¹³ (twice) formed mainly three multivalent configurations: Chain III+II, CIV+I and CV. — The autoradiographic study indicated an abnormal, precocious spiralization pattern for the chromatin in CIV+I primary spermatocytes. These cells, occurring together with the CIII+II and CV configurations in recognizable groups, usually descending from single spermatogonial stem cells, are delayed through meiotic prophase. Both delay and disturbed chromosome spiralization in these cells are attributed to the uniform association of the univalent (I) chromosome 1¹³ with the sex chromosomes during pachytene. Primary spermatocytes of the CIV+I configuration and those carrying a CV take longer to develop from metaphase I into secondary spermatocytes than does the CIII+II type. — In T70H tertiary trisomics with a similar chromosome imbalance, the majority of primary spermatocytes degenerates during the diakinesis-metaphase I stages of meiosis. Fertility is low in contrast to the translocation trisomics. Comparison between the two types leads to the conclusion, that trisomy per se reduces the size of the testes and that the univalent containing CIV+I primary spermatocytes, contrary to the almost uniformly 1¹³ univalent carrying spermatocytes of the T70H tertiary trisomics are rescued by the neighbouring CIII+II and CV carrying cells to form normal secondary spermatocytes and morphologically normal sperm.