Nanoscale dynamics and localization of single endogenous mRNAs in stress granules.

Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy. First, we developed a methodology for tracking single mRNAs within SGs, revealing that although mRNAs in SGs are mainly stationary (∼40%), they also move in a confined (∼25%) or freely diffusing (∼35%) manner. Second, the super-resolution localization microscopy showed that the mRNAs in SGs are heterogeneously distributed and partially form high-density clusters. Third, we simultaneously performed single mRNA tracking and super-resolution microscopy in SGs, demonstrating that single mRNA trajectories are mainly found around high-density clusters. Finally, a quantitative analysis of mRNA localization and dynamics during stress removal was conducted using live super-resolution imaging and single-molecule tracking. These results suggest that SGs have a highly organized structure that enables dynamic regulation of the mRNAs at the nanoscale, which is responsible for the ordered formation and the wide variety of functions of SGs.

A search for acrolein scavengers among food components.

Brain stroke is a major cause of being bedridden for elderly people, and preventing stroke is important for maintaining quality of life (QOL). Acrolein is a highly reactive aldehyde and causes tissue damage during stroke. Decreasing acrolein toxicity ameliorates tissue injury during brain stroke. In this study, we tried to identify food components which decrease acrolein toxicity. We found that 2-furanmethanethiol, cysteine methyl and ethyl esters, alliin, lysine and taurine decreased acrolein toxicity. These compounds neutralized acrolein by direct interaction. However, the interaction between acrolein and taurine was not so strong. Approximately 30 mM taurine was necessary to interact with 10 µM acrolein, and 2 g/kg taurine was necessary to decrease the size of mouse brain infarction. Taurine also slightly increased polyamine contents, which are involved in decrease in the acrolein toxicity. Mitochondrial potential damage by acrolein was also protected by taurine. Our results indicate that daily intake of foods containing 2-furanmethanethiol, cysteine methyl and ethyl esters, alliin, lysine and taurine may prevent severe injury in brain stroke and improve the quality of life for elderly people.

Transient Receptor Potential Ankyrin 1 (TRPA1) Channel Mediates Acrolein Cytotoxicity in Human Lung Cancer Cells.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective ion channel implicated in thermosensation and inflammatory pain. It has been reported that expression of the TRPA1 channel is induced by cigarette smoke extract. Acrolein found in cigarette smoke is highly toxic and known as an agonist of the TRPA1 channel. However, the role of TRPA1 in the cytotoxicity of acrolein remains unclear. Here, we investigated whether the TRPA1 channel is involved in the cytotoxicity of acrolein in human lung cancer A549 cells. The IC50 of acrolein in A549 cells was 25 µM, and acrolein toxicity increased in a concentration- and time-dependent manner. When the effect of acrolein on TRPA1 expression was examined, the expression of TRPA1 in A549 cells was increased by treatment with 50 µM acrolein for 24 h or 500 µM acrolein for 30 min. AP-1, a transcription factor, was activated in the cells treated with 50 µM acrolein for 24 h, while induction of NF-κB and HIF-1α was observed in the cells treated with 500 µM acrolein for 30 min. These results suggest that acrolein induces TRPA1 expression by activating these transcription factors. Overexpression of TRPA1 in A549 cells increased acrolein sensitivity and the level of protein-conjugated acrolein (PC-Acro), while knockdown of TRPA1 in A549 cells or treatment with a TRPA1 antagonist caused tolerance to acrolein. These findings suggest that acrolein induces the TRPA1 channel and that an increase in TRPA1 expression promotes the cytotoxicity of acrolein.

Higher serum alkaline phosphatase value indicates the need for bone mineral density testing in non-metastatic prostate cancer patients undergoing androgen deprivation therapy.

PURPOSE: Osteoporosis is a well-known adverse effect of androgen deprivation therapy for prostate cancer. This study aimed to reveal the factors associated with the diagnosis of osteoporosis in prostate cancer patients undergoing androgen deprivation therapy. METHODS: This retrospective cross-sectional study included 106 prostate cancer patients treated with androgen deprivation therapy. Patients with bone metastasis at the initiation of androgen deprivation therapy and those with castration-resistant prostate cancer were excluded. Bone mineral density was measured at the lumbar spine and femoral neck using dual-energy X-ray absorptiometry. Osteoporosis was defined as bone mineral density equal to or below either -2.5 SD or 70% of the mean in young adults. The association between clinicopathological variables and bone mineral density or diagnosis of osteoporosis was investigated. RESULTS: Thirty-six (34%) patients were found to have osteoporosis. The incidence of osteoporosis increased in a stepwise manner depending on the duration of androgen deprivation therapy. Multivariate logistic regression analysis identified a longer duration of androgen deprivation therapy (months, odd's ratio = 1.017, P = 0.006), lower body mass index (kg/m2, odd's ratio = 0.801, P = 0.005) and higher serum alkaline phosphatase value (U/l, odd's ratio 1.007, P = 0.014) as the factors independently associated with the diagnosis of osteoporosis. Eleven out of 50 (22%), 14 out of 35 (40%) and 11 out of 20 patients (55%) were osteoporotic in the patients with serum alkaline phosphatase values <238 U/l, 238-322 U/l and >322 U/l, respectively (P = 0.022). CONCLUSIONS: Osteoporosis is common in prostate cancer patients undergoing androgen deprivation therapy; furthermore, its incidence increases depending on the duration of androgen deprivation therapy. Bone mineral density testing should be considered for all patients on androgen deprivation therapy, especially for those with a lower body mass index and higher serum alkaline phosphatase value.

The immunosenescence-related factor DOCK11 is involved in secondary immune responses of B cells.

BACKGROUND: Memory B cells are an antigen-experienced B-cell population with the ability to rapidly differentiate into antibody-producing cells by recall responses. We recently found that dedicator of cytokinesis 11 (DOCK11) contributes to the expansion of antigen-specific populations among germinal center B cells upon immunization. In comparison, limited information is available on the contribution of DOCK11 to secondary humoral immune responses. RESULTS: In this study, effects of the DOCK11 deficiency in B cells were examined on secondary immune responses to protein antigen. The lack of DOCK11 in B cells resulted in the impaired induction of antibody-producing cells upon secondary immunization with protein antigen. DOCK11 was dispensable for the recall responses of antigen-experienced B cells, as demonstrated by the comparable induction of antibody-producing cells in mice given transfer of antigen-experienced B cells with no DOCK11 expression. Instead, the lack of DOCK11 in B cells resulted in the impaired secondary immune responses in a B cell-extrinsic manner, which was recovered by the adoptive transfer of cognate T cells. CONCLUSIONS: We addressed that intrinsic and extrinsic effects of DOCK11 expression in B cells may contribute to secondary humoral immune responses in manner of the induction of cognate T-cell help.

Alkaline Stress Causes Changes in Polyamine Biosynthesis in Thermus thermophilus.

An extreme thermophile, Thermus thermophilus, produces 16 different polyamines including long-chain and branched-chain polyamines. The composition and content of polyamines in the thermophile cells change not only with growth temperature but also with pH changes. In particular, cell growth decreased greatly at alkaline medium together with significant changes in the composition and content of polyamines. The amounts of tetraamines (spermine and its homologs) markedly decreased at alkaline pH. Thus, we knocked out the speE gene, which is involved in the biosynthesis of tetraamines, and changes of composition of polyamines with pH changes in the mutant cells were studied. Cell growth in the ΔspeE strain was decreased compared with that of the wild-type strain for all pHs, suggesting that tetraamines are important for cell proliferation. Interestingly, the amount of spermidine decreased and that of putrescine increased in wild-type cells at elevated pH, although T. thermophilus lacks a putrescine synthesizing pathway. In addition, polyamines possessing a diaminobutane moiety, such as spermine, decreased greatly at high pH. We assessed whether the speB gene encoding aminopropylagmatine ureohydrolase (TtSpeB) is directly involved in the synthesis of putrescine. The catalytic assay of the purified enzyme indicated that TtSpeB accepts agmatine as its substrate and produces putrescine due to the change in substrate specificity at high pH. These results suggest that pH stress was exacerbated upon intracellular depletion of polyamines possessing a diaminobutane moiety induced by unusual changes in polyamine biosynthesis under high pH conditions.

Polyamines regulate gene expression by stimulating translation of histone acetyltransferase mRNAs.

Polyamines regulate gene expression in Escherichia coli by translationally stimulating mRNAs encoding global transcription factors. In this study, we focused on histone acetylation, one of the mechanisms of epigenetic regulation of gene expression, to attempt to clarify the role of polyamines in the regulation of gene expression in eukaryotes. We found that activities of histone acetyltransferases in both the nucleus and cytoplasm decreased significantly in polyamine-reduced mouse mammary carcinoma FM3A cells. Although protein levels of histones H3 and H4 did not change in control and polyamine-reduced cells, acetylation of histones H3 and H4 was greatly decreased in the polyamine-reduced cells. Next, we used control and polyamine-reduced cells to identify histone acetyltransferases whose synthesis is stimulated by polyamines. We found that polyamines stimulate the translation of histone acetyltransferases GCN5 and HAT1. Accordingly, GCN5- and HAT1-catalyzed acetylation of specific lysine residues on histones H3 and H4 was stimulated by polyamines. Consistent with these findings, transcription of genes required for cell proliferation was enhanced by polyamines. These results indicate that polyamines regulate gene expression by enhancing the expression of the histone acetyltransferases GCN5 and HAT1 at the level of translation. Mechanistically, polyamines enhanced the interaction of microRNA-7648-5p (miR-7648-5p) with the 5'-UTR of GCN5 mRNA, resulting in stimulation of translation due to the destabilization of the double-stranded RNA (dsRNA) between the 5'-UTR and the ORF of GCN5 mRNA. Because HAT1 mRNA has a short 5'-UTR, polyamines may enhance initiation complex formation directly on this mRNA.

Translational Regulation of Clock Genes BMAL1 and REV-ERBα by Polyamines.

Polyamines stimulate the synthesis of specific proteins at the level of translation, and the genes encoding these proteins are termed as the "polyamine modulon". The circadian clock generates daily rhythms in mammalian physiology and behavior. We investigated the role of polyamines in the circadian rhythm using control and polyamine-reduced NIH3T3 cells. The intracellular polyamines exhibited a rhythm with a period of about 24 h. In the polyamine-reduced NIH3T3 cells, the circadian period of circadian clock genes was lengthened and the synthesis of BMAL1 and REV-ERBα was significantly reduced at the translation level. Thus, the mechanism of polyamine stimulation of these protein syntheses was analyzed using NIH3T3 cells transiently transfected with genes encoding enhanced green fluorescent protein (EGFP) fusion mRNA with normal or mutated 5'-untranslated region (5'-UTR) of Bmal1 or Rev-erbα mRNA. It was found that polyamines stimulated BMAL1 and REV-ERBα synthesis through the enhancement of ribosomal shunting during the ribosome shunting within the 5'-UTR of mRNAs. Accordingly, the genes encoding Bmal1 and Rev-erbα were identified as the members of "polyamine modulon", and these two proteins are significantly involved in the circadian rhythm control.

Cytotoxic Mechanism of Excess Polyamines Functions through Translational Repression of Specific Proteins Encoded by Polyamine Modulon.

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine-Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.

Pyruvate secreted from patient-derived cancer-associated fibroblasts supports survival of primary lymphoma cells.

Cancer-associated fibroblasts (CAF) are a key component in the tumor microenvironment and play functional roles in tumor metastasis and resistance to chemotherapies. We have previously reported that CAF isolated from lymphoma samples increase anaerobic glycolysis and decrease intracellular production of reactive oxygen species, promoting the survival of tumor cells. Herein, we analyzed the mechanisms underlying this support of tumor-cell survival by CAF. As direct contact between lymphoma cells and CAF was not indispensable to survival support, we identified that the humoral factor pyruvate was significantly secreted by CAF. Moreover, survival of lymphoma cells was promoted by the presence of pyruvate, and this promotion was canceled by inhibition of monocarboxylate transporters. Metabolome analysis of lymphoma cells in coculture with CAF demonstrated that intermediates in the citric acid cycle were significantly increased, indicating that tumor cells produced energy by aerobic metabolism. These findings indicate that energy production in lymphoma cells is regulated in coordination not only with anaerobic glycolysis, but also with aerobic metabolism termed the reverse-Warburg effect, involving the secretion of pyruvate from CAF resulting in increased use of the citric acid cycle in lymphoma cells.

Protective Effects of Brain Infarction by N-Acetylcysteine Derivatives.

BACKGROUND AND PURPOSE: We recently found that acrolein (CH2=CH-CHO) is more strongly involved in brain infarction compared with reactive oxygen species. In this study, we looked for acrolein scavengers with less side effects. METHODS: Photochemically induced thrombosis model mice were prepared by injection of Rose Bengal. Effects of N-acetylcysteine (NAC) derivatives on brain infarction were evaluated using the public domain National Institutes of Health image program. RESULTS: NAC, NAC ethyl ester, and NAC benzyl ester (150 mg/kg) were administered intraperitoneally at the time of induction of ischemia, or these NAC derivatives (50 mg/kg) were administered 3× at 24-h intervals before induction of ischemia and 1 more administration at the time of induction of ischemia. The size of brain infarction decreased in the order NAC benzyl ester>NAC ethyl ester>NAC in both experimental conditions. Detoxification of acrolein occurred through conjugation of acrolein with glutathione, which was catalyzed by glutathione S-transferases, rather than direct conjugation between acrolein and NAC derivatives. The level of glutathione S-transferases at the locus of brain infarction was in the order of administration of NAC benzyl ester>NAC ethyl ester>NAC>no NAC derivatives, suggesting that NAC derivatives stabilize glutathione S-transferases. CONCLUSIONS: The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione S-transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.

Involvement of Zizimin2/3 in the age-related defect of peritoneal B-1a cells as a source of anti-bacterial IgM.

Zizimin2 (Ziz2), also known as dedicator of cytokinesis 11 (DOCK11), is a guanine nucleotide exchange factor that is predominantly expressed in lymphoid tissues. Recent findings demonstrated that Ziz2 is involved in the development of B cells, including germinal centre B cells and marginal zone B cells. However, limited information is currently available on the roles of Ziz2 in B-1 cells, a B-cell subset that resides in body cavities and contributes to protection against foreign pathogens in a T-cell-independent manner. We herein show that Ziz2 and its widely expressed isoform Ziz3 (also known as DOCK10) may be involved in defective production of anti-bacterial IgM by aged B-1a cells, a CD5+ subset of B-1 cells. Natural IgM against typical bacterial epitopes was defectively produced by peritoneal B-1a cells from aged mice. The down-regulation of Ziz2/3 in B-1a cells appeared to be responsible for this defective IgM production, as demonstrated by Ziz2/3 double-knockout mice. Mechanistically, lower levels of basal AKT phosphorylation did not allow for the differentiation of Ziz2/3-deficient B-1a cells into plasma cells. Defective production of anti-bacterial IgM was not fully rescued by immunization, resulting in slightly weaker protection in Ziz2/3-deficient mice. Thus, the down-regulation of Ziz2/3 in B-1a cells may at least partly account for defective protection in aged mice.

Fenton reaction-induced renal carcinogenesis in Mutyh-deficient mice exhibits less chromosomal aberrations than the rat model.

Oxidative stress including iron excess has been associated with carcinogenesis. The level of 8-oxoguanine, a major oxidatively modified base in DNA, is maintained very low by three distinct enzymes, encoded by OGG1, MUTYH and MTH1. Germline biallelic inactivation of MUTYH represents a familial cancer syndrome called MUTYH-associated polyposis. Here, we used Mutyh-deficient mice to evaluate renal carcinogenesis induced by ferric nitrilotriacetate (Fe-NTA). Although the C57BL/6 background is cancer-resistant, a repeated intraperitoneal administration of Fe-NTA induced a high incidence of renal cell carcinoma (RCC; 26.7%) in Mutyh-deficient mice in comparison to wild-type mice (7.1%). Fe-NTA treatment also induced renal malignant lymphoma, which did not occur without the Fe-NTA treatment in both the genotypes. Renal tumor-free survival after Fe-NTA treatment was marginally different (P = 0.157) between the two genotypes. Array-based comparative genome hybridization analyses revealed, in RCC, the loss of heterozygosity in chromosomes 4 and 12 without p16INKA inactivation; these results were confirmed by a methylation analysis and showed no significant difference between the genotypes. Lymphomas showed a preference for genomic amplifications. Dlk1 inactivation by promoter methylation may be involved in carcinogenesis in both tumors. Fe-NTA-induced murine RCCs revealed significantly less genomic aberrations than those in rats, demonstrating a marked species difference.

SPIB is a novel prognostic factor in diffuse large B-cell lymphoma that mediates apoptosis via the PI3K-AKT pathway.

Although the clinical outcomes of diffuse large B-cell lymphoma (DLBCL) have improved in the immunochemotherapy era, approximately one-third of patients develop intractable disease. To improve clinical outcomes for these patients, it is important to identify those with poor prognosis prior to initial treatment in order to select optimal therapies. Here, we investigated the clinical and biological significance of SPIB, an Ets family transcription factor linked to lymphomagenesis, in DLBCL. We classified 134 DLBCL patients into SPIB negative (n = 108) or SPIB positive (n = 26) groups by immunohistochemical staining. SPIB positive patients had a significantly worse treatment response and poor prognosis compared with SPIB negative patients. Multivariate analysis for patient survival indicated that SPIB expression was an independent poor prognostic factor for both progression free survival (PFS) and overall survival (OS) (PFS, hazard ratio [HR] 2.65, 95% confidence interval [CI] 1.31-5.33, P = 0.006; OS, HR 3.56, 95% CI 1.43-8.91, P = 0.007). Subsequent analyses of the roles of SPIB expression in DLBCL pathogenesis revealed that SPIB expression in lymphoma cells resulted in resistance to the BH3-mimetic ABT-263 and contributed to apoptosis resistance via the PI3K-AKT pathway. The inhibition of AKT phosphorylation re-sensitized SPIB expressing lymphoma cells to ABT-263-induced cell death. Together, our data indicate that SPIB expression is a clinically novel poor prognostic factor in DLBCL that contributes to treatment resistance, at least in part, through an anti-apoptotic mechanism.

Polyamine stimulation of eEF1A synthesis based on the unusual position of a complementary sequence to 18S rRNA in eEF1A mRNA.

It is thought that Shine-Dalgarno-like sequences, which exhibit complementarity to the nucleotide sequences at the 3'-end of 18S rRNA, are not present in eukaryotic mRNAs. However, complementary sequences consisting of more than 5 nucleotides to the 3'-end of 18S rRNA, i.e., a CR sequence, are present at -17 to -32 upstream from the initiation codon AUG in 18 mRNAs involved in protein synthesis except eEF1A mRNA. Thus, effects of the CR sequence in mRNAs and polyamines on protein synthesis were examined using control and polyamine-reduced FM3A and NIH3T3 cells. Polyamines did not stimulate protein synthesis encoded by 18 mRNAs possessing a normal CR sequence. When the CR sequence was deleted, protein synthetic activities decreased to less than 70% of intact mRNAs. In eEF1A mRNA, the CR sequence was located at -33 to -39 upstream from the initiation codon AUG, and polyamines stimulated eEF1A synthesis about threefold. When the CR sequence was shifted to -22 to -28 upstream from the AUG, eEF1A synthesis increased in polyamine-reduced cells and the degree of polyamine stimulation decreased greatly. The results indicate that the CR sequence exists in many eukaryotic mRNAs, and the location of a CR sequence in mRNAs influences polyamine stimulation of protein synthesis.

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance in Escherichia coli.

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance were studied using a polyamine biosynthesis and uptake deficient Escherichia coli KK3131 transformed with pACYC184 containing potFGHI or puuP. Putrescine uptake activity of E. coli KK3131 transformed with pACYC184-PotFGHI was higher than that of E. coli 3131 transformed with pACYC-PuuP when cells were cultured in the absence of putrescine. Putrescine uptake by PotFGHI was both ATP and membrane potential dependent, while that by PuuP was membrane potential dependent. Feedback inhibition by polyamines occurred at the PotFGHI uptake system but not at the PuuP uptake system. Expression of PuuP was reduced in the presence of PuuR, a negative regulator for PuuP, and expression of PuuR was positively regulated by glucose, which reduces the level of cAMP. The complex of cAMP and CRP (cAMP receptor protein) inhibited the expression of PuuR in the absence of glucose. Thus, the growth rate of E. coli KK3131 in the presence of both 0.4% (22.2 mM) glucose and 10 mM putrescine was in the order of cells transformed with pACYC-PotFGHI > pACYC-PuuP > pACYC-PuuP + PuuR, which was parallel with the polyamine content in cells. The results indicate that PotFGHI is necessary for rapid cell growth in the presence of glucose as an energy source. When glucose in medium was depleted, however, PuuP was absolutely necessary for cell growth in the presence of putrescine, because accumulation of putrescine to a high level by PuuP was necessary for utilization of putrescine as an energy source.

Caldomycin, a new guanidopolyamine produced by a novel agmatine homocoupling enzyme involved in homospermidine biosynthesis.

An extreme thermophilic bacterium, Thermus thermophilus produces more than 20 unusual polyamines, but their biosynthetic pathways, including homospermidine, are not yet fully understood. Two types of homospermidine synthases have been identified in plants and bacteria, which use spermidine and putrescine or two molecules of putrescine as substrates. However, homospermidine synthases with such substrate specificity have not been identified in T. thermophilus. Here we identified a novel agmatine homocoupling enzyme that is involved in homospermidine biosynthesis in T. thermophilus. The reaction mechanism is different from that of a previously described homospermidine synthase, and involves conjugation of two molecules of agmatine, which produces a diamidino derivative of homospermidine (caldomycin) as an immediate precursor of homospermidine. We conclude that there is a homospermidine biosynthetic pathway from agmatine via caldomycin synthase followed by ureohydrolase in T. thermophilus. Furthermore, it is shown that caldomycin is a novel compound existing in nature.

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.

Inactivation of GAPDH as one mechanism of acrolein toxicity.

We have recently reported that acrolein is more toxic than reactive oxygen species. Thus, the mechanism of cell toxicity by acrolein was studied using mouse mammary carcinoma FM3A cells. Acrolein-conjugated proteins were separated by gel electrophoresis with subsequent determination of their amino acid sequence, and it was found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was one of the major acrolein-conjugated proteins in cells. Acrolein interacted with cysteine-150 at the active site of GAPDH, and also with cysteine-282. When cells were treated with 8 µM acrolein, the activity of acrolein-conjugated GAPDH was greatly reduced, and the ATP content in cells was thus significantly reduced. In addition, it was shown that acrolein-conjugated GAPDH translocated to the nucleus, and the level of acetylated GAPDH and the number of TUNEL positive cells was increased, indicating that cell death is enhanced by acrolein-conjugated GAPDH. Inhibition of cell growth by acrolein was partially reversed when the cDNA encoding GAPDH was transformed into cells. These results indicate that inactivation of GAPDH is one mechanism that underlies cell toxicity caused by acrolein.

Spermidine is required for morphogenesis in the human pathogenic fungus, Penicillium marneffei.

Penicillium marneffei is a thermally dimorphic fungus that is a highly significant pathogen of immune compromised persons living or having traveled in Southeast Asia. When cultured at 25°C, the wild-type strain of P. marneffei exhibits a mycelial morphology that is marked by the development of specialized structures bearing conidia. Incubation of the wild type at 37°C, however, promotes the development of a yeast form that divides by fission. Development of the yeast morphology in vivo appears to be requisite for pathogenesis. In a prior study using Agrobacterium-mediated transformation for random mutagenesis via T-DNA integration, we generated a morphological mutant (strain I6) defective in conidiation. The T-DNA insertion site in strain I6 was determined to be within the gene encoding S-adenosylmethionine decarboxylase (sadA), an enzyme critical to spermidine biosynthesis. In the present study, we demonstrated that strain I6 was able to grow on rich media in either the mold or yeast forms at 25°C and 37°C, respectively. However, reduced growth of strain I6 was observed on minimal medium at either temperature. In addition, strain I6 produced mycelia with impaired conidiation on minimal medium at 25°C. Supplementation of minimal medium with spermidine restored the ability of strain I6 to produce conidia at 25°C and promoted yeast development at 37°C. Moreover, conidia of strain I6 exhibited poor germination frequencies in the absence of this polyamine. All three of these processes (conidiogenesis, germination, and growth) were reinstated in strain I6 by complementation of the partially deleted of sadA gene by ectopic insertion of an intact wild-type copy. These results augment prior observations that spermidine biosynthesis is essential to normal growth, conidiogenesis, spore germination, and dimorphism in a variety of fungi. Given the presumption that P. marneffei infections are initiated following inhalation of conidia, and that pathogenesis is dependent upon yeast development, this study further suggests that the spermidine biosynthetic pathway may serve as a potential target for combating infections by this medically important fungus.