Genetic Control of Immune Responses to a Synthetic Fimbrial Antigen of Actinobacillus actinomycetemcomitans

The incidence of infection by Actinobacillus actinomycetemcomitans, one of the important pathogens in human periodontal diseases, has been reported to be associated with racial background and genetic factors. We attempted to determine the genetic regulation of immune responses to A. actinomycetemcomitans fimbriae, an attachment factor, using various inbred strains of mice. For this purpose, we synthesized an oligopeptide antigen using the amino acid sequence of the fimbriae and conjugated this antigen to branched lysine polymer resin beads. After immunization with the synthetic A. actinomycetemcomitans fimbrial antigen, serum antibody levels and the delayed-type hypersensitivity (DTH) reaction to the antigen were measured by enzyme-linked immunosorbent assay (ELISA) and footpad swelling responses, respectively. The strains of mice found to be high-IgG responders to the antigen were B10.HTT, B10.RIII, B10A (5R) and B10.S (9R). These results indicate that mice with E_β^s: E_α^k, E_β^r: E_α^r and E_β^b: E_α^k respond strongly to the synthetic peptide. All of the high-IgG responders showed a high DTH response. A cell transfer experiment confirmed that CD4 T cells mediated with a DTH response to the synthetic peptide. Thus, the results of this study demonstrate that the immune responses to A. actinomycetemcomitans fimbriae are genetically controlled.     

High Mortality of Disseminated Non-Tuberculous Mycobacterial Infection in HIV-Infected Patients in the Antiretroviral Therapy Era

Background

Little information is available on the mortality and risk factors associated with death in disseminated non-tuberculous mycobacterial infection (dNTM) in HIV-infected patients in the ART-era.

Methods

In a single-center study, HIV-infected dNTM with positive NTM culture from sterile sites between 2000 and 2013 were analysed. The clinical characteristics at commencement of anti-mycobacterial treatment (baseline) were compared between those who survived and died.

Results

Twenty-four patients were analyzed. [The median CD4 27/μL (range 2–185)]. Mycobacterium avium and M. intracellulare accounted for 20 (83%) and 3 (13%) of isolated NTM. NTM bacteremia was diagnosed in 15 (63%) patients. Seven (29%) patients died, and NTM bacteremia was significantly associated with mortality (p = 0.022). The baseline CD4 count was significantly lower in the non-survivors than the survivors (median 7/μL versus 49, p = 0.034). Concomitant AIDS-defining diseases or malignancies were not associated with mortality. Immune-reconstitution syndrome (IRS) occurred to 19 (79%) patients (8 paradoxical and 11 unmasking), and prognosis tended to be better in unmasking-IRS than the other patients (n = 13) (p = 0.078). Patients with paradoxical-IRS had marginally lower CD4 count and higher frequency of bacteremia than those with unmasking-IRS (p = 0.051, and 0.059). Treatment with systemic corticosteroids was applied in 63% and 55% of patients with paradoxical and unmasking-IRS, respectively.

Conclusion

dNTM in HIV-infected patients resulted in high mortality even in the ART-era. NTM bacteremia and low CD4 count were risk factors for death, whereas patients presented with unmasking-IRS had marginally better prognosis. IRS occurred in 79% of the patients, suggesting difficulty in the management of dNTM.     

Nucleotide sequence of the gene for Escherichia coli ribosomal protein S15 (rpsO)

Summary The nucleotide sequence of the ribosomal protein gene rpsO (S15) and its flanking region were determined. The amino acid sequence of S15 protein deduced from the nucleotide sequence is in good agreement with the published amino acid sequence with one exception. The nucleotide sequence shows two probable promoter sites about 100 nucleotides upstream from the initiation codon (AUG) of rpsO. Inspection of the sequence also revealed structural homology between the distal part of rpsO and the reported S15 binding region in 16S rRNA.     

Embryonic and morphological development of larvae and juveniles of the Amberjack,Seriola dumerili

Embryonic and morphological development of larvae and juveniles of the amberjack,Seriola dumerili Risso, are described using specimens raised at Yaeyama Station (Ishigaki Island, Okinawa Pref.), Japan Sea Farming Association. The specimens obtained from brood fish (3 females, 3 males) were treated with gonadotropin and spawned on 6th of April 1987. The eggs of amberjack are pelagic, spherical in shape and 1.01–1.17 mm in diameter. The yolk is roughly segmented and has a single oil globule 0.22–0.24 mm in diameter. The perivitelline space is narrow. During development, a few melanophores and no xanthophores were observed on yolk. Hatching took place 35 hrs. 15 min. after spawning out at temperatures 23.1–23.7°C. The newly hatched larvae were 2.84–3.04mm in TL with 27 (13+14) myomeres and an oil globule anteriorly situated beyond the head. 3 days after hatching 4.00 mm TL, the mouth opened. 10 days after hatching 4.26 mm TL, small denticles appeared on the margin of the upper jaw and there were 1 anterior and 2 posterior preopecular spines. At 5.96mm TL, notochord was slightly flexed. Caudal, dorsal and anal fins with rudiments of rays appeared at 8.00 mm TL. The specific numbers of all fin rays and spines were obtained in a juvenile 9.60 mm TL. In a juvenile 34.25 mm TL, 54 days after hatching, the characteristic brown band of amberjack had appeared on head. Some notable changes in relative growth were observed at 5 mm and 15 mm in TL.     

Synthesis and evaluation of in vivo anti‐hypothermic effect of all stereoisomers of the thyrotropin‐releasing hormone mimetic: Rovatirelin Hydrate

We discovered the orally active thyrotropin‐releasing hormone (TRH) mimetic: (4S,5S)‐5‐methyl‐N‐{(2S)‐1‐[(2R)‐2‐methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide 1 (rovatirelin). The central nervous system (CNS) effect of rovatirelin after intravenous (iv) administration is 100‐fold higher than that of TRH. As 1 has four asymmetric carbons in its molecule, there are 16 stereoisomers. We synthesized and evaluated the anti‐hypothermic effect of all stereoisomers of 1 , which has the (4S),(5S),(2S),(2R) configuration from the N‐terminus to the C‐terminus, in order to clarify the structure?activity relationship (SAR) of stereoisomers. The (4R),(5R),(2R),(2S)‐isomer 16 did not show any anti‐hypothermic effect. Only the (4S),(5S),(2S),(2S)‐isomer 10 , which has the (2S)‐2‐methylpyrrolidine moiety at the C‐terminus showed the anti‐hypothermic effect similar to 1 . Stereoisomers, which have the (5R) configuration of the oxazolidinone at the N‐terminus and the (2R) configuration at the middle‐part, showed a much lower anti‐hypothermic effect than that of 1 . On the other hand, stereoisomers, which have the (4R) configuration of the oxazolidinone at the N‐terminus or the (2S) configuration of the C‐terminus, have little influence on the anti‐hypothermic effect.     

New application of firefly luciferase--it can catalyze the enantioselective thioester formation of 2-arylpropanoic acid

We introduce a new application of firefly luciferase (EC 1.13.12.7). The firefly luciferases belong to a large superfamily that includes rat liver long-chain acyl-CoA synthetase (LACS1). LACS1 is the enzyme that is involved in the deracemization process of 2-arylpropanoic acid and catalyzes the enantioselective thioester formation of R-acids. Based on the similarity of the reaction mechanisms and the sequences between firefly luciferase and LACS1, we predicted that firefly luciferase also has thioesterification activity toward 2-arylpropanoic acid. From an investigation using three kinds of luciferases from North American firefly and Japanese fireflies, we have confirmed that these luciferases exhibit an enantioselective thioester formation activity and the R-form is transformed to a thioester in preference to the S-form in the presence of ATP, Mg(2+), and CoASH. The enantiomeric excesses of unreacted recovered acid and thioester were determined by chiral phase HPLC analysis and the resulting 2-arylpropanoyl-CoAs were identified by high resolution mass spectroscopy. The K(m) and k(cat) values of thermostable luciferase from Luciola lateralis (LUC-H) toward ketoprofen were determined as 0.22 mM and 0.11 s(-1), respectively. The affinity of ketoprofen was almost the same of d-luciferin. In addition, the calculated E-value toward ketoprofen was approximately 20. These results suggest that LUC-H could catalyze the kinetic resolution of 2-arylpropanoic acid efficiently and would be a new option for the preparation of optically active 2-substituted carboxylic acids.     

Effects of carbohydrate substrate on the vegetative mycelial growth of an ectomycorrhizal mushroom, <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>, isolated from <Emphasis Type="Italic">Quercus</Emphasis>

We studied the characteristics of the utilization of carbohydrate substrates and the production of those hydrolyzing enzymes of the Tricholoma matsutake J-1 strain isolated from hardwood (Quercus sp.). In the culture medium, 5% glucose inhibited mycelial growth. The growth inhibition rate was remarkable in the Z-1 strain from softwood (Pinus densiflora) compared with that of the J-1 strain from hardwood. α-Amylase production varied with starches from different origins in contrast to mycelial growth. The range of the effect of 0.5%–15% soluble starch on vegetative mycelial growth was also investigated. The optimal concentration for mycelial growth was 15% for the J-1 strain but 10% for the Z-1 strain. Mycelial growth of the J-1 strain was strongly inhibited in PMML medium containing Sunpeal-CP prepared from sulfite pulp softwood waste, but that of the Z-1 strain was not inhibited by Sunpeal-CP. Moreover, mycelial growth of the J-1 strain from Quercus sp. dramatically decreased with the addition of CNF-HWSF (hot water-soluble fractions from corn fiber) to the PMML and PDL medium. However, inhibition by CNF-HWSF was not shown in the Z-1 strain from P. densiflora.     

Design,synthesis and evaluation of novel zwitterionic compounds as PPARα/γ dual agonists (1)

We describe here the design, syntheses and structure–activity relationships (SAR) of novel zwitterionic compounds as non-thiazolidinedion (TZD) based peroxisome proliferator activated receptor (PPAR) α/γ dual agonists. We commenced the medicinal research with compound 1 originated by Eli Lilly, which was reported to possess PPAR α/γ dual agonist activity. We incorporated an amine linker and optimized it on the nitrogen of the linker, thereby envisioning the enhancement of the PPAR α/γ dual agonist activity together with altering the physicochemical properties. As a result, we could generate compounds showing the PPAR α/γ dual activity, especially among which compound 22e had a franylmethyl group on the linker and 2,6-dimethyl phenyl ring at the carboxylic acid head group furnishing a highly potent dual agonist activity, together with a great glucose lowering effect. Moreover, it remedied the lipid profile, that is, triglyceride without body weight gain in the db/db mice model.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.