Synthesis and characterization of 3,13- and 2,13-octadecadienyl compounds for identification of the sex pheromone secreted by a clearwing moth, Nokona pernix

Several geometrical isomers of 3,13- and 2,13-octadecadien-1-ols and their acetates were synthesized starting from 1,8-octanediol or 1,9-nonanediol utilizing acetylene coupling reactions. In addition to commercially available compounds, all geometrical isomers of each dienyl compound were analyzed by NMR and GC-MS to accumulate chemical data for studies of sex pheromones secreted from clearwing moths classified into the family Sesiidae of Lepidoptera. Although acetoxy derivatives of the 3,13- and 2,13-dienes showed almost the same mass spectra, the alcohols were distinguished by comparing the relative intensities of [M-18](+) at m/z 248, indicating direct differentiation of the two positional isomers without derivatization. Furthermore, each geometrical isomer eluted from a high-polar GC column with a different retention time. Base on these data, a pheromone gland extract of a sesiid moth, Nokona pernix, was analyzed by GC-EAD and GC-MS, and two EAG-active components were identified, viz., the (3E,13Z)- and (3Z,13Z)-isomers of 3,13-octadecadien-1-ol in a ratio of 9:1. In the field, the synthetic compounds mixed in 9:1 ratio attracted N. pernix males well, while a single component scarcely attracted the males. The number of attracted males peaked in the middle of June, and a small second peak was observed in August.     

Induction of differential T-cell epitope by plain- and liposome-coupled antigen

The T-cell receptors of CD4(+) T lymphocytes recognize immunogenic peptide sequences bound within the groove of MHC class II molecules, and the peptides that bind to these molecules are known to share common structural motifs. For example, OVA(323-339), an I-A(d)-binding peptide, involves a motif of the I-A(d) peptide-binding groove. In the present study, OVA peptides of up to 26-mer were sequentially synthesized and screened, and two additional I-A(d) binding OVA peptides, OVA(20-43) and OVA(264-286), were found to stimulate CD4(+) T cells of OVA-immune BALB/c mice. OVA(20-43) involved structural motifs of the I-A(d) peptide-binding groove, while OVA(264-286) did not. The ability of these three I-A(d) binding OVA peptides to induce antigen-specific cytokine production was compared among CD4(+) T cells of mice immunized either with alum-adsorbed OVA (OVA-alum) or OVA chemically coupled to the surface of liposome (OVA-liposome). CD4(+) T cells of mice immunized with OVA-alum produced more cytokines when stimulated with OVA(264-286) than with OVA(323-339), while CD4(+) T cells of mice immunized with OVA-liposome conjugates produced more cytokines when stimulated with OVA(323-339) than with OVA(264-286). OVA(20-43) induced production of comparable levels of cytokines in mice immunized either with OVA-alum or OVA-liposome. Confocal laser scanning microscopic analysis demonstrated that chemically coupled OVA and liposomes were colocalized in APCs until OVA received processing. Three-dimensional structural analysis demonstrated that both OVA(264-286) and OVA(323-339) were present on the surface of OVA, but OVA(20-43) was not. These results suggested that the chemical coupling of OVA to liposome affected antigen processing in APCs and thus resulted in the induction of differential T-cell epitopes as compared with those induced by plain OVA.     

Lipofuscin-like fluorophores originated from malondialdehyde

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of post-mitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. In the present mini-review, I provide a comprehensive summary of fluorescent adducts originated from malondialdehyde (MDA).     

Analysis of HIV-1 sequences before and after co-infecting syphilis

Increasing syphilis incidence among men who have sex with men (MSM) has been reported. The index case was a human immunodeficiency virus type 1 (HIV-1)-positive MSM who presented coincidentally with the secondary syphilis and a rebound of plasma viral load after complete suppression of HIV-1 (below 50 copies/ml) for 13 months with potent antiretroviral therapy (PART), suggesting a possibility of HIV-1 superinfection. We analyzed HIV-1 sequences before and after syphilis in four HIV-1-positive patients including the index case to explore drug resistance mutations (DRMs) and a possibility of HIV-1 superinfection. There were patients who obtained DRMs around syphilis infection but no evidence of HIV-1 superinfection was obtained. Our results underline the importance of strict adherence to PART.     

New approach to in situ quantification of ovarian gene expression in rat using a laser microdissection technique: relationship between follicle types and regulation of inhibin-α and cytochrome P450aromatase genes in the rat ovary

The recently developed laser microdissection (LMD) technique makes it possible to quantify local gene expression in the target cells of various tissues. Using the LMD technique, this study aimed at comparing the amounts of mRNAs encoding the inhibin-α subunit and cytochrome P450 aromatase (P450_arom) in granulosa cells between preantral and antral follicles in immature rat ovaries. Serial frozen sections of the ovaries from 24-day-old female Wistar rats were made and 30 healthy preantral (100–200 μm maximum diameter) and ten healthy antral ( > 300 μm maximum diameter) follicles were selected in each ovary based on morphological examinations, including immunohistochemistry for inhibin-α, in sections adjacent to those used for LMD. The amounts of mRNAs encoding inhibin-α subunit and P450_arom were quantified by real-time polymerase chain reaction (PCR). While the amount of P450_arom mRNA in the granulosa cell layers from the antral follicles was about 12-times higher than that in the preantral follicles, no difference in the amount of inhibin-α mRNA was found between these two types of follicles. Thus, the LMD technique allowed the in situ quantification of gene expression in the ovary and revealed that each granulosa cell expresses a stable amount of inhibin-α subunit mRNA independently of antral formation in immature rat ovaries.     

Polymeric grape-seed procyanidins, but not monomeric catechins and oligomeric procyanidins, impair degranulation and membrane ruffling in RBL-2H3 cells

Grape-seed proanthocyanidins (GSPs) are catechin polymers that are predicted to form helices in their global minimum-energy conformation and to have a mean degree of polymerization of seven (mDP = 7). The highly polymerized GSP-H fraction (mDP = 10) was found to impair degranulation in RBL-2H3 cells after stimulation with an antigen (Ag) and treatment with the Ca-ATPase inhibitor thapsigargin (Tg). In addition, GSP-H affected actin cytoskeleton and inhibited membrane ruffling in these cells, resulting in the suppression of exocytosis. By contrast, monomeric epicatechin, the dimeric procyanidins PA-1, PA-2, and PB-2, and the oligomerized GSP-L (mDP = 3) had no effect on membrane ruffling and degranulation. These findings indicate that the molecular size and length of GSP-H are needed for the inhibition of membrane ruffling and degranulation in RBL-2H3 mast-cell lines.     

Targeted serum glycoproteomics for the discovery of lung cancer‐associated glycosylation disorders using lectin‐coupled ProteinChip arrays

To screen for glycoproteins showing aberrant sialylation patterns in sera of cancer patients and apply such information for biomarker identification, we performed SELDI‐TOF MS analysis coupled with lectin‐coupled ProteinChip arrays (Jacalin or SNA) using sera obtained from lung cancer patients and control individuals. Our approach consisted of three processes (i) removal of 14 abundant proteins in serum, (ii) enrichment of glycoproteins with lectin‐coupled ProteinChip arrays, and (iii) SELDI‐TOF MS analysis with acidic glycoprotein‐compatible matrix. We identified 41 protein peaks showing significant differences (p<0.05) in the peak levels between the cancer and control groups using the Jacalin‐ and SNA‐ProteinChips. Among them, we identified loss of Neu5Ac (α2,6) Gal/GalNAc structure in apolipoprotein C‐III (apoC‐III) in cancer patients through subsequent MALDI‐QIT‐TOF MS/MS. Furthermore, subsequent validation experiments using an additional set of 60 lung adenocarcinoma patients and 30 normal controls demonstrated that there is a higher frequency of serum apoC‐III with loss of α2,6‐linkage Neu5Ac residues in lung cancer patients compared to controls. Our results have demonstrated that lectin‐coupled ProteinChip technology allows the high‐throughput and specific recognition of cancer‐associated aberrant glycosylations, and implied a possibility of its applicability to studies on other diseases.     

Human Neuroglobin Functions as an Oxidative Stress-responsive Sensor for Neuroprotection

Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. The mechanism underlying this function is only partly understood. Here, we report that human Ngb exists in lipid rafts only during oxidative stress and that lipid rafts are crucial for neuroprotection by Ngb. The ferrous oxygen-bound form of Ngb, which exists under normoxia, is converted to the ferric bis-His conformation during oxidative stress, inducing large tertiary structural changes. We clarified that ferric bis-His Ngb, but not ferrous ligand-bound Ngb, specifically binds to flotillin-1, a lipid raft microdomain-associated protein, as well as to α-subunits of heterotrimeric G proteins (Gα(i/o)). Moreover, we found that human ferric bis-His Ngb acts as a guanine nucleotide dissociation inhibitor for Gα(i/o) that has been modified by oxidative stress. In addition, our data shows that Ngb inhibits the decrease in cAMP concentration that occurs under oxidative stress, leading to protection against cell death. Furthermore, by using a mutated Ngb protein that cannot form the bis-His conformation, we demonstrate that the oxidative stress-induced structural changes of human Ngb are essential for its neuroprotective activity.