Persistence of gene expression changes in stomach mucosae induced by short-term N-methyl-N'-nitro-N-nitrosoguanidine treatment and their presence in stomach cancers

Cancers induced by different carcinogens show distinct expression profiles. In addition to the specific alterations of tumor-related genes induced by specific carcinogens, it is possible that some initial responses induced by a carcinogen could persist for long periods and are consistently present in the cancers induced. We have analyzed the initial responses in the rat pyloric mucosae after treatment for 2 weeks with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Gene expression was monitored 1 day, 2 weeks and 4 weeks after MNNG treatment by oligonucleotide microarray analysis. Of the differentially expressed genes showing greater than three-fold difference 1 day after MNNG treatment, 143 and 26 genes were up- and down-regulated, respectively, in MNNG-induced stomach cancers. Among these genes, 25 and 6 genes were up- and down-regulated, respectively, in the histologically normal pyloric mucosae, even 4 weeks after cessation of MNNG treatment. Among the up-regulated genes, many genes involved in tissue remodeling (Spi15, Serpine1 and Fst) and cellular growth (Bdnf, Ros1 and Fgf10) were present. The six down-regulated genes included TGF-beta-inducible early growth response gene. These findings demonstrate that some expression changes induced by MNNG persist for a prolonged period and are present in cancers. Persistent expression changes are considered to be important for prediction of past carcinogen exposure, and could provide a molecular environment favorable for malignant transformation.     

Differential Anti-APOBEC3G Activity of HIV-1 Vif Proteins Derived from Different Subtypes

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.     

N-terminal mutational analysis of the interaction between growth-blocking peptide (GBP) and receptor of insect immune cells

GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The 1H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core beta-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn2. Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.     

Dominant role of thyrotropin-releasing hormone in the hypothalamic-pituitary-thyroid axis

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the beta isoforms of TH receptors (TRbeta KO), or both (double KO). TRbeta knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.     

Magnetic characterization of Daphnia resting eggs

This study characterized the magnetic materials found within Daphnia resting eggs by measuring static magnetization with a superconducting quantum interference device (SQUID) magnetometer, after forming two types of conditions, each of which consists of zero-field cooling (ZFC) and field cooling (FC). Magnetic ions, such as Fe(3+), contained in Daphnia resting eggs existed as (1) paramagnetic and superparamagnetic particles, demonstrated by a magnetization and temperature dependence of the magnetic moments under an applied magnetic field after ZFC and FC, and (2) ferromagnetic particles with definite magnetic moments, the content of which was estimated to be very low, demonstrated by the Moskowitz test. Conventionally, biomagnets have been directly detected by transmission electron microscopes (TEM). As demonstrated in this study, it is possible to nondestructively detect small biomagnets by magnetization measurement, especially after two types of ZFC and FC. This nondestructive method can be applied in detecting biomagnets in complex biological organisms.     

GABAergic inhibition regulates developmental synapse elimination in the cerebellum

Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.     

Synthesis of the 3-sulfates of S-acyl glutathione conjugated bile acids and their biotransformation by a rat liver cytosolic fraction

The 3-sulfates of the S-acyl glutathione (GSH) conjugates of five natural bile acids (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic) were synthesized as reference standards in order to investigate their possible formation by a rat liver cytosolic fraction. Their structures were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion-trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. This method was used to determine whether the 3-sulfates of the bile acid-GSH conjugates (BA-GSH) were formed when BA-GSH were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate had been added. The S-acyl linkage was rapidly hydrolyzed to form the unconjugated bile acid. A little sulfation of the GSH conjugates occurred, but greater sulfation at C-3 of the liberated bile acid occurred. Sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus GSH conjugates of bile acids as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes.     

Structural Confirmation of a Unique Carotenoid Lactoside,P457, in Symbiodinium sp. Strain nbrc 104787 Isolated from a Sea Anemone and its Distribution in Dinoflagellates and Various Marine Organisms

The molecular structure of the carotenoid lactoside P457, (3S,5R,6R,3′S,5′R,6′S)‐13′‐cis‐5,6‐epoxy‐3′,5′‐dihydroxy‐3‐(β‐d ‐galactosyl‐(1→4)‐β‐d ‐glucosyl)oxy‐6′,7′‐didehydro‐5,6,7,8,5′,6′‐hexahydro‐β,β‐caroten‐20‐al, was confirmed by spectroscopic methods using Symbiodinium sp. strain NBRC 104787 cells isolated from a sea anemone. Among various algae, cyanobacteria, land plants, and marine invertebrates, the distribution of this unique diglycosyl carotenoid was restricted to free‐living peridinin‐containing dinoflagellates and marine invertebrates that harbor peridinin‐containing zooxanthellae. Neoxanthin appeared to be a common precursor for biosynthesis of peridinin and P457, although neoxanthin was not found in peridinin‐containing dinoflagellates. Fucoxanthin‐containing dinoflagellates did not possess peridinin or P457; green dinoflagellates, which contain chlorophyll a and b, did not contain peridinin, fucoxanthin, or P457; and no unicellular algae containing both peridinin and P457, other than peridinin‐containing dinoflagellates, have been observed. Therefore, the biosynthetic pathways for peridinin and P457 may have been coestablished during the evolution of dinoflagellates after the host heterotrophic eukaryotic microorganism formed a symbiotic association with red alga that does not contain peridinin or P457.     

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SG4502 with acquired LA-polymerizing activity

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1_SG and PhaC2_SG). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1_SG to evaluate the potential of the resulting protein as a “thermostable LPE”. The mutated PhaC1_SG [PhaC1_SG(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1_SG(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).     

Involvement of T6 pili in biofilm formation by serotype M6 Streptococcus pyogenes

The group A streptococcus (GAS) Streptococcus pyogenes is known to cause self-limiting purulent infections in humans. The role of GAS pili in host cell adhesion and biofilm formation is likely fundamental in early colonization. Pilus genes are found in the FCT (fibronectin-binding protein, collagen-binding protein, and trypsin-resistant antigen) genomic region, which has been classified into nine subtypes based on the diversity of gene content and nucleotide sequence. Several epidemiological studies have indicated that FCT type 1 strains, including serotype M6, produce large amounts of monospecies biofilm in vitro. We examined the direct involvement of pili in biofilm formation by serotype M6 clinical isolates. In the majority of tested strains, deletion of the tee6 gene encoding pilus shaft protein T6 compromised the ability to form biofilm on an abiotic surface. Deletion of the fctX and srtB genes, which encode pilus ancillary protein and class C pilus-associated sortase, respectively, also decreased biofilm formation by a representative strain. Unexpectedly, these mutant strains showed increased bacterial aggregation compared with that of the wild-type strain. When the entire FCT type 1 pilus region was ectopically expressed in serotype M1 strain SF370, biofilm formation was promoted and autoaggregation was inhibited. These findings indicate that assembled FCT type 1 pili contribute to biofilm formation and also function as attenuators of bacterial aggregation. Taken together, our results show the potential role of FCT type 1 pili in the pathogenesis of GAS infections.