The Structure of Leucanicidin,a Novel Insecticidal Macrolide Produced by Streptomyces halstedii

( – )-Invictolide [(3R,5R,6S,1′R)-3,5-dimethyl-6-(1′-methylbutyl)-tetrahydro-2H-pyran-2-one] was synthesized in 16 steps from 2-methylpentanal.     

Reactivation of immune responses against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> by boosting with the CpG oligomer in aged mice primarily vaccinated with <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

Background

Mycobacterium bovis bacillus Calmette Guérin (BCG) vaccine, which has been inoculated to more than one billion people world-wide, has significant effect in preventing tuberculous meningitis and miliary tuberculosis (TB) in neonate and early childhood. However, BCG fails to adequately protect against pulmonary TB and reactivation of latent infections in adults. To overcome this problem, adequate booster is urgently desired in adult who received prior BCG vaccination, and appropriate animal models that substitute human cases would be highly valuable for further experimentation.

Findings

The booster effect of the synthesized CpG oligomer (Oligo-B) on aged mice which had been primarily vaccinated with BCG at the age of 4-week old. The specific Th1 type reaction, production of interferon-γ, in response to TB antigens, purified protein derivatives (PPD) and protection against challenge with Mycobacterium tuberculosis (MTB) H₃₇Rv decreased with increasing age and were not observed in 89-week old mice. In order to rejuvenate the Th1 type response against PPD and protection activity against MTB infection, Oligo-B, which is known to augment Th1 responses, was administered as a booster to 81-90-week old mice (late 50’s in human equivalent) vaccinated with BCG at 4-week old. The boosting with Oligo-B increased the number of CD4⁺ CD44^high CD62L^high, central memory type T cell. Furthermore, the Oligo-B boosting rejuvenated the ability of mice to protect against infection with MTB H₃₇Rv.

Conclusions

Th1-adjuvant CpG oligo DNA, such as Oligo-B, may be a promising booster when coupled with BCG priming.

     

Bis (2,4,6-Tribromophenyl) Phosphorochloridate: A New Type of Condensing Reagent in Oligonucleotide Synthesis

Abstract

The detailed study of internucleotidic bond formation by the use of bis(2,4,6-tribromophenyl) phosphorochloridate (TBP) is described. The ³¹P NMR analysis suggested that the TBP-mediated condensation proceeded via a reactive unsymmetrical pyrophosphate intermediate, which was readily activated in the presence of 3-nitro-1,2,4-triazole to form internucleotidic bond.     

Synthesis of Adenosine,Guanosine and Cytidine Monomer Building Units Bearing the [[2-(Methylthio)-phenyl]thio]methyl (MPTM) Group as The 2′-Hydroxyl Protecting Group

Abstract

A new 2′-hydroxyl protecting group, [[2-(methylthio)phenyl]thio]methyl (MPTM), was introduced into the 2′-position of adenosine, guanosine and cytidine building units required for the synthesis of oligoribonucleotides.     

Stereocontrolled Synthesis of Dithymidine Phosphorothioates by Use of a Functionalized 5′- Protecting Group Bearing an Imidazole Residue†

Abstract

Diastereoselective formation of internucleotidic phosphorothioate triester bonds was achieved by use of 3-(imidazol-1-ylmethyl)-4′,4″-dimethoxytrityl (IDTr) as a 5′-hydroxyl protecting group in the phosphotriester approach. After removal of all the protecting groups, stereochemistry of the major product was determined as the Spconfiguration by enzymatic digestion.

     

The Cellular and Molecular Basis of Bitter Tastant-Induced Bronchodilation

Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca²⁺ signals, as revealed in cultured ASM cells, to activate large-conductance Ca²⁺-activated K⁺ channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca²⁺ events nor alter spontaneous local Ca²⁺ transients. Interestingly, they increase global intracellular [Ca²⁺]_i, although to a much lower level than bronchoconstrictors. We show that these Ca²⁺ changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ [PLCβ]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca²⁺]_i induced by bronchoconstrictors, and this lowering of the [Ca²⁺]_i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca²⁺ channels (VDCCs), resulting in reversal in [Ca²⁺]_i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca²⁺ signaling pathways via Gβγ to increase [Ca²⁺]_i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca²⁺]_i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.     

High-throughput genotyping in citrus accessions using an SNP genotyping array

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.     

Identification of domains on the fusion (F) protein trimer that influence the hemagglutinin-neuraminidase specificity of the f protein in mediating cell-cell fusion

For most paramyxoviruses, virus type-specific interaction between fusion (F) protein and attachment protein (hemagglutinin-neuraminidase [HN], hemagglutinin [H], or glycoprotein [G]) is a prerequisite for mediating virus-cell fusion and cell-cell fusion. Our previous cell-cell fusion assay using the chimeric F proteins of human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41) suggested that the middle region of the HPIV2 F protein contains the site(s) that determines its specificity for the HPIV2 HN protein. In the present study, we further investigated the sites of the F protein that could be critical for determining the HN protein specificity. By analyzing the reported structure of the F protein of parainfluenza virus 5 (PIV5), we found that four major domains (M1, M2, M3, and M4) and five minor domains (A to E) in the middle region of the PIV5 F protein were exposed on the trimer surface. We then replaced these domains with the SV41 F counterparts individually or in combination and examined whether the resulting chimeras could mediate cell-cell fusion when coexpressed with the SV41 HN protein. The results showed that a chimera designated M(1+2), which harbored SV41 F-derived domains M1 and M2, mediated cell-cell fusion with the coexpressed SV41 HN protein, suggesting that these domains are involved in determining the HN protein specificity. Intriguingly, another chimera which harbored the SV41 F-derived domain B in addition to domains M1 and M2 showed increased specificity for the SV41 HN protein compared to that of M(1+2), although it was capable of mediating cell-cell fusion by itself.     

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates

Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products.     

p125/Sec23-interacting protein (Sec23ip) is required for spermiogenesis

p125/Sec23ip is a phospholipase A(1)-like protein that interacts with Sec23, a coat component of COPII vesicles that bud from endoplasmic reticulum exit sites. To understand its physiological function, we produced p125 knockout mice. The p125 knockout mice grew normally, but males were subfertile. Sperm from p125-deficient mice had round heads and lacked the acrosome, an organelle containing the enzymes responsible for fertilization. p125 was found to be expressed at stages I-XII of spermatogenesis, similar to the expression pattern of proteins involved in acrosome biogenesis. These results suggest that p125 plays an important role in spermiogenesis.