Potent antibody-mediated neutralization and evolution of antigenic escape variants of simian immunodeficiency virus strain SIVmac239 in vivo

Here, we describe the evolution of antigenic escape variants in a rhesus macaque that developed unusually high neutralizing antibody titers to SIVmac239. By 42 weeks postinfection, 50% neutralization of SIVmac239 was achieved with plasma dilutions of 1:1,000. Testing of purified immunoglobulin confirmed that the neutralizing activity was antibody mediated. Despite the potency of the neutralizing antibody response, the animal displayed a typical viral load profile and progressed to terminal AIDS with a normal time course. Viral envelope sequences from week 16 and week 42 plasma contained an excess of nonsynonymous substitutions, predominantly in V1 and V4, including individual sites with ratios of nonsynonymous to synonymous substitution rates (dN/dS) highly suggestive of strong positive selection. Recombinant viruses encoding envelope sequences isolated from these time points remained resistant to neutralization by all longitudinal plasma samples, revealing the failure of the animal to mount secondary responses to the escaped variants. Substitutions at two sites with significant dN/dS values, one in V1 and one in V4, were independently sufficient to confer nearly complete resistance to neutralization. Substitutions at three additional sites, one in V4 and two in gp41, conferred moderate to high levels of resistance when tested individually. All the amino acid changes leading to escape resulted from single nucleotide substitutions. The observation that antigenic escape resulted from individual, single amino acid replacements at sites well separated in current structural models of Env indicates that the virus can utilize multiple independent pathways to rapidly achieve similar levels of resistance.     

Solar-simulated light-exposed benzo[a]pyrene induces phosphorylation of histone H2AX

Polycyclic aromatic hydrocarbons (PAHs), wide-spread mutagenic and carcinogenic environmental pollutants, are consistently exposed to sunlight in the environment. The exposure causes structural change, resulting in the generation of a variety of photomodified products having different bioactivities compared with the parent compounds. In this study, we found that benzo[a]pyrene (BaP) exposed to solar-simulated light (SSL)-induced phosphorylation of histone H2AX (γ-H2AX), which was recently identified as an early event after the induction of DNA double strand breaks (DSBs). Although BaP itself did not produce γ-H2AX, SSL-exposed BaP significantly generated γ-H2AX depending on the period of exposure. Furthermore, we revealed that reactive oxygen species produced by the SSL-exposed BaP mainly contributed to the generation of γ-H2AX. The appearance of γ-H2AX means the induction of the most serious form of DNA damage, DSBs, suggesting the potential risk of carcinogenesis.     

Sugar Chips immobilized with synthetic sulfated disaccharides of heparin/heparan sulfate partial structure

Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate-protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or heparan sulfate responsible for binding were identified.     

L-Tyrosine production by Polyauxotrophic Mutants of Corynebacterium glutamicum

Polyauxotrophic mutants of Corynebacterium glutamicum which have additional requirements to L-phenylalanine were derived from L-tyrosine producing strains of phenylalanine auxotrophs, C. glutamicum KY 9189 and C. glutamicum KY 10233, and screened for L-tyrosine production. The increase of L-tyrosine production was noted in many auxotrophic mutants derived from both strains. Especially some double auxotrophs which require phenylalanine and purine, phenylalanine and histidine, or phenylalanine and cysteine produced significantly higher amounts of L-tyrosine compared to the parents, A phenylalanine and purine double auxotrophic strain LM–96 produced L-tyrosine at a concentration of 15.1 mg per ml in the medium containing 20% sucrose. L-Tyrosine production by the strain decreased at high concentrations of L-phenylalanine.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

Streptococcus cremoris was cultivated for 7 days at 30°C in sterilized skim milk or in the sterilized 10% solution of dry skim milk. This skim milk culture was divided into precipitate and supernatant by centrifugation. The absorbancy at 280 mμ of the supernatant prepared from the skim milk culture of S. cremoris was higher than that of the control supernatant.

Casein prepared from the skim milk culture of S. cremoris was less hydrolyzed by rennet than control casein at pH 7.0.

According to the free boundary electrophoretic analysis of the treated casein in m/10 veronal buffer of pH 8.5 containing urea, α-casein seemed to be hydrolyzed by S. cremoris but β-casein did with more difficulty.     

Purification of 7-Keto-8-aminopelargonic Acid-7,8-Diaminopelargonic Acid Aminotransferase,an Enzym Involved in Biotin Biosynthesis,from Brevibacterium divaricatum

Vitamin B₆ is synthesized by green Cytisus scoparius callus and green Phellodendron amurense callus cultured on Linsmaier and Skoog Agar-medium with 10^?5m of ±-naphthaleneacetic acid (NAA) and 10^?6 m of 6-benzyladenine (BA). Even when thiamine and inositol were omitted from this medium, the growth and vitamin B₆ content of Cytisus scoparius callus did not change. Vitamin B₆ contents of clones of the calluses varied and were unstable during long-term subculture. Clonal selection was repeated to obtain stable strains with high vitamin B₆ content, and the vitamin B₆ content of one strain of green Cytisus scoparius callus became 4-times higher than that of the green leaves.     

Fermentation of C14-Labeled Cellobiose by Cellulomonas fimi

Crude cell-free extracts from Cellulomonas fimi contain cellobiose phosphorylase which cleaves cellobiose into glucose and glucose-1-phosphate in the presence of inorganic phosphate. With the aid of this enzyme, two samples of C¹⁴-cellobiose labeled in reducing or non-reducing glucosyl moiety were prepared from uniformly labeled C¹⁴-glucose or C¹⁴-glucose-1-phosphate as substrate, respectively. The labeled preparations have been shown to be radiochemically pure. Analyses of the anaerobic fermentation products from C¹⁴-cellobiose by resting cell suspensions showed that both glucose moieties were fermented almost equivalently. However, relatively small differences in specific activities of the products revealed that significantly larger amounts of formic acid and smaller amounts of acetic acid were produced from the reducing glucose moiety than from the other half of the molecule. Succinic and lactic acids appeared to be produced almost equally from both moieties.     

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective

To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs).

Methods

This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009.

Results

Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset.

Conclusion

Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”     

Matrix-Embedded Osteocytes Regulate Mobilization of Hematopoietic Stem/Progenitor Cells

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Therapeutic effect of risedronate on cancellous and cortical bone in ovariectomized osteopenic rats: a comparison with the effects of alfacalcidol.

The purpose of the present study was to compare the therapeutic effects of risedronate (RIS) and alfacalcidol (ALF) on cancellous and cortical bone in ovariectomized osteopenic rats. Forty-two female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into six groups: the sham-operated control (Sham) group, and five ovariectomized groups: treated with vehicle, RIS (0.1, 1.0, or 2.5 mg/kg, p.o., daily), and ALF (0.5 microg/kg, p.o., daily). Treatment was started 6 weeks after surgery and continued for 6 weeks. Evaluation at 12 weeks after surgery revealed that ovariectomy (OVX) decreased the cancellous bone volume/total tissue volume (BV/TV) of the proximal tibial metaphysis as a result of an increase of the bone formation rate/bone surface (BFR/BS), BFR/BV, and eroded surface (ES/BS), while having no effect on the cortical area (Ct Ar) of the tibial diaphysis. OVX also decreased the maximum load of the femoral distal metaphysis, while having no effect on any mechanical property parameters of the femoral diaphysis. RIS (at all the doses) increased the BV/TV relative to the value in the OVX-Vehicle group, but the value was not restored to that observed in the Sham group. The effects of RIS (1.0 mg/kg and 2.5 mg/kg) were similar, and greater than those of RIS (0.1 mg/kg). ALF also increased the BV/TV relative to the OVX-Vehicle group, but the value was not restored to that observed in the Sham group, similar to the results of RIS (1.0 mg/kg and 2.5 mg/kg) treatment. The alterations of the structural parameters induced by RIS (at the doses) were attributable to suppression of the increase of ES/BS, BFR/BS, and BFR/BV. The alterations of the structural parameters induced by ALF were attributable to suppression of the increase of ES/BS and attenuation of the increase of BFR/BV, while the BFR/BS was maintained. ALF also increased the Ct Ar to beyond the value observed in the Sham group. RIS (at all the doses) had no effect on the mechanical properties of the femoral distal metaphysis, whereas ALF prevented the loss of the maximum load of the femoral distal metaphysis. Thus, the results of the present study show differential effects of RIS and ALF on cancellous and cortical bone in ovariectomized osteopenic rats.