Identification of the primary electron donor in PS II of the Chl d-dominated cyanobacterium Acaryochloris marina

The primary electron donor of photosystem (PS) II in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina was confirmed by delayed fluorescence (DF) and further proved by pigment contents of cells grown under several light intensities. The DF was found only in the Chl a region, identical to Synechocystis sp. PCC 6803, and disappeared following heat treatment. Pigment analyses indicated that at least two Chl a molecules were present per each two pheophytin a molecules, and these Chl a molecules are assigned to P(D1) and P(D2). These findings clearly indicate that Chl a is required for water oxidation in PS II.     

Novel biological function of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger

We have found that N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H(2)O(2)) under physiological conditions. Close investigation of this finding revealed that NANA was oxidized by an equimolar amount of H(2)O(2) to provide its decarboxylated product, 4-(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (ADOA). To date, there have been little data on this reaction, and its physiological significance has not been discussed. Examining the detoxification of H(2)O(2) in cultured cells with NANA, we were able to confirm that the cell death caused by H(2)O(2) was suppressed by NANA in a dose-dependent manner. These results revealed a novel role for NANA as a reactive oxygen scavenger. It is known that terminal NANA residues are removed by neuraminidase and that free NANA molecules are recycled or degraded by enzymes. We propose that released monomeric NANA is the potent defense molecule against oxidative damage.     

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.     

Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant Mycobacterium bovis bacille Calmette-Guérin expressing human immunodeficiency virus type 1 Gag

Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.     

Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.     

Phosphorylation of candida glabrata ATP-binding cassette transporter Cdr1p regulates drug efflux activity and ATPase stability

Fungal ATP-binding cassette transporter regulation was investigated using Candida glabrata Cdr1p and Pdh1p expressed in Saccharomyces cerevisiae. Rephosphorylation of Pdh1p and Cdr1p was protein kinase A inhibitor-sensitive but responded differentially to Tpk isoforms, stressors, and glucose concentration. Cdr1p Ser(307), which borders the nucleotide binding domain 1 ABC signature motif, and Ser(484), near the membrane, were dephosphorylated on glucose depletion and independently rephosphorylated during glucose exposure or under stress. The S484A enzyme retained half the wild type ATPase activity without affecting azole resistance, but the S307A enzyme was unstable to plasma membrane isolation. Studies of pump function suggested conformational interaction between Ser(484) and Ser(307). An S307A/S484A double mutant, which failed to efflux the Cdr1p substrate rhodamine 6G, had a fluconazole susceptibility 4-fold greater than the Cdr1p expressing strain, twice that of the S307A mutant, but 64-fold less than the control null strain. Stable intragenic suppressors indicative of homodimer nucleotide binding domain 1-nucleotide binding domain 1 interactions partially restored rhodamine 6G pumping and increased fluconazole and rhodamine 6G resistance in the S307A/S484A mutant. Nucleotide binding domain 1 of Cdr1p is a sensor of important physiological stimuli.     

Genetic coadaptation of the amylase gene system in Drosophila melanogaster: evidence for the selective advantage of the lowest AMY activity and of its epistatic genetic background

In natural populations of Drosophila melanogaster, an amylase isozyme with the lowest alpha-amylase activity (AMY(1,1)) is predominant. To evaluate the selective significance of AMY(1,1) and its regulatory factor(s), we examined selection experiments in laboratory populations on two distinct food environments. After 300 generations, AMY(1,1) became predominant (89%) in a glucose (a product of AMY)-rich environment, while an isozyme with higher alpha-amylase activity, AMY(1,6), became predominant (83%) in a starch (substrate)-rich environment. We found that the identical alleles of the amylase (Amy) gene, which encodes each of AMY(1,1) and AMY(1,6), were shared between the two populations in the different food environments, employing the nucleotide sequencing of the duplicated Amy genes. Nevertheless, AMY(1,6) homozygotes selected in the starch-rich environment had a twofold higher AMY enzyme activity than those selected in the glucose-rich environment, suggesting a coadaptation of the coding region and its regulatory factor(s) on the genetic background. Such a difference in AMY enzyme activity was not detected between AMY(1,1) homozygotes, suggesting that the effect of the genetic background is epistatic. Our results indicate that natural selection is working on the Amy gene system as a whole for flies to adapt to the various food environments of local populations.     

The role of ICOS in the CXCR5+ follicular B helper T cell maintenance in vivo

ICOS is a new member of the CD28 family of costimulatory molecules that is expressed on activated T cells. Its ligand B7RP-1 is constitutively expressed on B cells. Although the blockade of ICOS/B7RP-1 interaction inhibits T cell-dependent Ab production and germinal center formation, the mechanism remains unclear. We examined the contribution of ICOS/B7RP-1 to the generation of CXCR5+ follicular B helper T (T(FH)) cells in vivo, which preferentially migrate to the B cell zone where they provide cognate help to B cells. In the spleen, anti-B7RP-1 mAb-treated or ICOS-deficient mice showed substantially impaired development of CXCR5+ T(FH) cells and peanut agglutinin+ germinal center B cells in response to primary or secondary immunization with SRBC. Expression of CXCR5 on CD4+ T cells was associated with ICOS expression. Adoptive transfer experiments showed that the development of CXCR5+ T(FH) cells was enhanced by interaction with B cells, which was abrogated by anti-B7RP-1 mAb treatment. The development of CXCR5+ T(FH) cells in the lymph nodes was also inhibited by the anti-B7RP-1 mAb treatment. These results indicated that the ICOS/B7RP-1 interaction plays an essential role in the development of CXCR5+ T(FH) cells in vivo.