Four‐year successive rearing of Glyptotendipes tokunagai Sasa (Diptera: Chironomidae) under laboratory condition

The dipteran Chironomidae have been commonly used as water quality indicators and toxicity test organisms. Two chironomids, Chironomus riparius Meigan and C. tentans Fabricius, are standard test organisms for toxicity (OECD), but their distribution is limited in Korea. The purpose of this study was to establish a Korean native chironomid species as a toxicity test organism. Glyptotendipes tokunagai Sasa, distributed widely in Korean streams, was selected and reared successively under laboratory conditions for over 30 generations over 4 years. Four G. tokunagai egg masses were collected from the Jungrang stream in Seoul, Korea in April 2007 and introduced to the laboratory for rearing. Room temperature (23.5 ± 3.2°C), larval and adult rearing cages, mating cages, and larval food were appropriately modified from conventional chironomid rearing methods. The number of eggs in an egg mass, hatching rate, and adult body sizes (head width, thorax width, wing width and length, and body length) were monitored every generation. As a result, the number of eggs in an egg mass decreased rapidly in early generations, and then tended to stabilize after the fourth generation (p < 0.05). The mean hatching rate was higher than 75% in all generations. The gender ratio (no. of females/total no. of adults) was 0.24–0.52. The adult body size became significantly reduced in the initial three generations and tended to be stabilized in the following generations (p < 0.05), although it depended on food supply and larval density. This is the first case of chironomid domestication in Korea that has been successfully reared longer than 4 years under laboratory conditions. This reared population of G. tokunagai can be used for various environmental studies such as bioassays, ecological risk assessments, and environmental monitoring.     

On the sensitivity of forward scattering patterns from bacterial colonies to media composition

Morphology of colonies is important for taxonomy and diagnostics in microbiology where the response to environmental factors is sensitive enough to support discrimination. In this research, we analyzed the forward scattering patterns of individual Escherichia coli K12 colonies when agar hardness and nutrition levels were varied from the control sample. As the agar concentration increased from 1.2% to 1.8%, the diameter of the forward scattering patterns also increased for the same experimental condition which reflects that the colony thickness at the apex is greater for increased agar concentrations. Regarding nutrition, increasing dextrose resulted in smaller mean colony diameters while the mean diameters of the colonies were proportional to the yeast extract concentration up to 0.5%. The result reveals that ±0.3% agar concentration from the control sample is sufficient to create variations in the scattering patterns. For nutrition –0.25% of yeast extract showed significant variations while +0.25% from control sample showed minimal variations. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Identification of gene expression signature modulated by nicotinamide in a mouse bladder cancer model

Background

Urinary bladder cancer is often a result of exposure to chemical carcinogens such as cigarette smoking. Because of histological similarity, chemically-induced rodent cancer model was largely used for human bladder cancer studies. Previous investigations have suggested that nicotinamide, water-soluble vitamin B3, may play a key role in cancer prevention through its activities in cellular repair. However, to date, evidence towards identifying the genetic alterations of nicotinamide in cancer prevention has not been provided. Here, we search for the molecular signatures of cancer prevention by nicotinamide using a N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced urinary bladder cancer model in mice.

Methodology/Principal Findings

Via microarray gene expression profiling of 20 mice and 233 human bladder samples, we performed various statistical analyses and immunohistochemical staining for validation. The expression patterns of 893 genes associated with nicotinamide activity in cancer prevention were identified by microarray data analysis. Gene network analyses of these 893 genes revealed that the Myc and its associated genes may be the most important regulator of bladder cancer prevention, and the gene expression signature correlated well with protein expression data. Comparison of gene expression between human and mouse revealed that BBN-induced mouse bladder cancers exhibited gene expression profiles that were more similar to those of invasive human bladder cancers than to those of non-invasive human bladder cancers.

Conclusions/Significance

This study demonstrates that nicotinamide plays an important role as a chemo-preventive and therapeutic agent in bladder cancer through the regulation of the Myc oncogenic signature. Nicotinamide may represent a promising therapeutic modality in patients with muscle-invasive bladder cancer.     

An engineered viral protease exhibiting substrate specificity for a polyglutamine stretch prevents polyglutamine-induced neuronal cell death

Background

Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington''s disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches.

Methodology/Principal Findings

Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2′, or P3′, but not substrates containing glutamine at the P2 or P1′ positions. To accommodate glutamine at P2 and P1′, key residues comprising the active sites of the S2 or S1′ pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3′. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent.

Conclusions/Significance

These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.     

The complete mitochondrial genome of the greater horseshoe bat subspecies, Rhinolophus ferrumequinum korai (Chiroptera: Rhinolophidae)

The total length of the mitogenome of Rhinolophus ferrumequinum korai is 16,839?bp with a total base composition of 31.8% A, 25.4% T, 28.7% C, and 14.0% G. The mitogenome consists of 13 protein-coding genes, 2 rRNA (12S and 16S RNA) genes, 22 tRNA genes, and 1 control region.     

Chaperone Stress 70 Protein (STCH) Binds and Regulates Two Acid/Base Transporters NBCe1-B and NHE1

Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96–440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na⁺-dependent pH_i recovery from NH₄⁺ pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na⁺/H⁺ exchanger (NHE)-dependent pH_i recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca²⁺ ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pH_i through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pH_i regulation at times of cellular stress by enhancing the recovery from intracellular acidification.     

Conservation and Variability in the Structure and Function of the Cas5d Endoribonuclease in the CRISPR-Mediated Microbial Immune System

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form an RNA-mediated microbial immune system against invading foreign genetic elements. Cas5 proteins constitute one of the most prevalent Cas protein families in CRISPR–Cas systems and are predicted to have RNA recognition motif (RRM) domains. Cas5d is a subtype I-C-specific Cas5 protein that can be divided into two distinct subgroups, one of which has extra C-terminal residues while the other contains a longer insertion in the middle of its N-terminal RRM domain. Here, we report crystal structures of Cas5d from Streptococcus pyogenes and Xanthomonas oryzae, which respectively represent the two Cas5d subgroups. Despite a common domain architecture consisting of an N-terminal RRM domain and a C-terminal β-sheet domain, the structural differences between the two Cas5d proteins are highlighted by the presence of a unique extended helical region protruding from the N-terminal RRM domain of X. oryzae Cas5d. We also demonstrate that Cas5d proteins possess not only specific endoribonuclease activity for CRISPR RNAs but also nonspecific double-stranded DNA binding affinity. These findings suggest that Cas5d may play multiple roles in CRISPR-mediated immunity. Furthermore, the specific RNA processing was also observed between S. pyogenes Cas5d protein and X. oryzae CRISPR RNA and vice versa. This cross-species activity of Cas5d provides a special opportunity for elucidating conserved features of the CRISPR RNA processing event.