Identification,characterization and expression profiling of cation-proton antiporter superfamily in Triticum aestivum L. and functional analysis of TaNHX4-B

The monovalent cation proton antiporter (CPA) superfamily comprises Na⁺/H⁺ exchanger (NHX), K⁺ efflux antiporter (KEA), and cation/H⁺ exchanger (CHX) family proteins, which play vital functions in plants. A total of 107 TaCPA proteins were identified in Triticum aestivum, and phylogenetically classified into 35 TaNHX, 24 TaKEA and 48 TaCHX proteins. These families had representatives derived from all three sub-genomes. TaKEA genes consisted of higher number of exons, followed by TaNHXs and TaCHXs. The occurrence of about 10 transmembrane regions and higher composition of helices and coils support their membrane-bound and hydrophobic nature. Diverse expression in various tissues and modulated expression under stress conditions suggested their role in development and in response to stress. Co-expression analyses revealed their complex interaction networks. Expression of TaNHX4-B.1 and TaNHX4-B.4 facilitated differential abiotic stress tolerance to Escherichia coli. Our study provides comprehensive information about CPA genes, which would be useful in their future functional characterization.     

Definition of Mafa-A and -B haplotypes in pedigreed cynomolgus macaques (Macaca fascicularis)

The major histocompatibility complex (MHC) class I B gene/allelic repertoire was investigated in a pedigreed population of cynomolgus macaques of mixed Indonesian/Malaysian origin. The Mafa-B alleles detected in this cohort are mostly specific for a given geographic area, and only a small number of alleles appears to be shared with other populations. This suggests the fast evolution of Mafa-B alleles due to adaptation to new environments. In contrast to humans, the B locus in Old World monkeys displays extensive copy number variation. The Mafa-B and previously defined -A gene combinations segregate in families and thus allowed the definition of extended haplotypes. In many cases it was possible to assign a particular Mafa-I allele to one of these Mafa-A/B haplotypes as well. The presence of a large number of stable haplotypes in this cohort of animals, which was pedigreed for up to eight generations, looks promising for developing discriminative MHC typing tools that are less cumbersome. Furthermore, the discovery of 53 unreported Mafa-B sequences expands the lexicon of alleles significantly, and may help in understanding the complex organisation of the macaque B region.     

Characterization of an aminopeptidase from Pseudozyma hubeiensis 31-B and potential applications

Yeast strain 31-B was isolated from the digestive juices of Nepenthes alata as an aminopeptidase producer and identified as Pseudozyma hubeiensis via morphological testing and comparative 26S ribosomal DNA-D1/D2 gene sequence analysis. Strain 31-B produced aminopeptidase as extracellular peptidase, but proteinase activity was not detected in the culture filtrate. The aminopeptidase from strain 31-B was purified from filtered culture medium by (NH₄)₂SO₄ precipitation and four column chromatography steps: Diethylaminoethyl (DEAE)-Toyopearl 650 M, Butyl-Toyopearl 650 M, hydroxylapatite, and Toyopearl HW-55. Sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded the purified enzyme as a single band with molecular mass 75.3 kDa. The optimum temperature and pH were approximately 40 °C and 8.0, respectively. The purified aminopeptidase preferentially hydrolyzed Leu-p-NA and its activity was inhibited by ethylenediaminetetraacetic acid. The isolated aminopeptidase reduced the bitterness of peptides generated from milk casein using a bacterial proteinase. These results show that the aminopeptidase produced by P. hubeiensis 31-B has potential application as a food additive in the dairy industry.     

Generation of the HLA-B35, -B5, -B16, and B15 groups of alleles studied by intron 1 and 2 sequence analysis

 HLA-B is the most polymorphic of the major histocompatibility complex classical class I loci. This polymorphism is mainly in exons 2 and 3, which code for the molecule’s α1 and α2 domains and include the antigenic peptide binding site. Recent studies have indicated that not only exons but also the intron 2 region may be involved in the generation of certain HLA-B alleles such as B ^* 3906 and B ^* 1522. To study the degree of intron 2 participation and the mechanisms that generate polymorphism at the HLA-B locus, intron 1 and 2 sequences from the HLA-B35, -B5, -B16 and -B15 groups of alleles were obtained. A group-specific intronic polymorphism was found: namely, B ^* 5301 shows intron 1 and 2 sequences identical to those found in all B35 alleles studied. On the other hand, B ^* 5101 and B ^* 52012 show the same intron 1 and 2 sequences and their intron 1 is the same as that found in the B35 group. This suggests that B5 and B35 groups of alleles may have arisen from a common ancestor. All known B16 alleles show the same introns 1 and 2, with the exception of B ^* 39061 and B ^* 39062, and all B15 alleles also bear the same introns 1 and 2, with the exception of B ^* 1522. Variability at intron 1 is more restricted than at intron 2, and the use of intron 1 for HLA-B allele phylogenetic analysis is better for grouping alleles of a postulated common origin. In conclusion, there is a remarkable conservation of intronic sequences within related HLA-B alleles, which probably reflects a common origin and perhaps a selective force avoiding DNA changes. Intronic sequences are also potentially useful to design DNA typing strategies. Received: 11 March 1997 / Revised: 29 May 1997     

Synthesis of d-phenylalanine oligopeptides catalyzed by alkaline d-peptidase from Bacillus cereus DF4-B

Synthesis of d-phenylalanine oligopeptides from d-phenylalanine methylester has been demonstrated by use of alkaline d-peptidase (ADP) from Bacillus cereus. An expression plasmid pKADP was constructed by placing the PCR-amplified ADP gene (adp) under the tac promoter of pKK223-3. Oligomerization of d-phenylalanine methylester by use of the purified ADP from the transformant Escherichia coli was investigated under several conditions. d-Phenylalanine dimer, (d-Phe)₂, and trimer, (d-Phe)₃, were produced in 25.4% and 8.6% yield, respectively, when 50 mM of the substrate was incubated for 8 h with ADP (2.0 U/ml and 0.4 U/ml, respectively) in 100 mM triethylamine–HCl (pH 11.5). Addition of dimethylsulfoxide to the reaction mixture resulted in the production of tetramer, (d-Phe)₄ in 6.7% yield with the decrease of the (d-Phe)₂ and (d-Phe)₃ production. This is the first study on the synthesis of d-phenylalanine oligomers by use of a d-stereospecific endopeptidase.     

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Vacuolar H⁺-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC₅₀ of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.     

Structural diversity and evolution of the N-terminal isoform-specific region of ecdysone receptor-A and -B1 isoforms in insects

Background

Species in the varied geographic topology of Taiwan underwent obvious demographic changes during glacial periods. Cinnamomum kanehirae has been exploited for timber and to obtain medicinal fungi for the past 100 years. Understanding anthropogenic factors influencing the demography of this species after the last glacial maximum (LGM) is critically important for the conservation of this species.

Results

Populations of C. kanehirae were classified into four geographic regions: northwestern (NW), west-central (WC), southwestern (SW), and southeastern (SE). In total, 113 individuals from 19 localities were sampled, and variations in the chalcone synthase gene (Chs) intron and leafy (Lfy) intron-2 sequences of nuclear DNA were examined in order to assess phylogeographic patterns, the timescales of demographic and evolutionary events, and recent anthropogenic effects. In total, 210 Chs and 170 Lfy sequences, which respectively constituted 36 and 35 haplotypes, were used for the analyses. Estimates of the migration rate (M) through time revealed a pattern of frequent gene flow during previous and the present interglacials. The isolation-by-distance test showed that there generally was no significant correlation between genetic and geographic distances. The level of among-region genetic differentiation was significant when comparing eastern to western populations. However, no significant among-region genetic differentiation was found in comparisons among the four geographic regions. Moreover, essentially no genetic structuring was found for the three regions west of the CMR. A fit of spatial range expansion was found for pooled and regional samples according to the non-significant values of the sum of squared deviations. Using the Bayesian skyline plot (BSP) method, a recent bottleneck after the LGM expansion was detected in both regional and pooled samples.

Conclusions

Common haplotype distributions among geographic regions and the relatively shallow genetic structuring displayed are the result of historical gene flows. Southward dispersals in an earlier time frame from the NW region and in a later time frame from the SE region were inferred. The BSP analysis suggested a postglacial expansion event. Recent trends, however, refer to a bottleneck due to human interventions observed for both pooled and regional C. kanehirae samples.     

Effect of Yeast Extract and Vitamin B12 on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B₁₂, biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B₁₂ alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

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.     

The germacranolides of Viguiera buddleiaeformis structures of budlein-A and -B

The structure of budlein-A, the main sesquiterpene lactone of Viguiera buddleiaeformis was established as the 8 angeloyl ester of 1 keto, 8-β, 14-dihydroxy germacra-2,4,11 (13)-trien-3, (10 β) oxido-6 α, 12-olide. Its structure and stereochemistry was determined by chemical and spectroscopic means. Budlein-B, found in the same plant as a minor constituent, is 8 α, 15-dihydroxygermacra-1 (10), 4, 11 (13)-trien-6 α, 12-olide.     

Polarized fluorescence measurements on ordered photosynthetic antenna complexes: Chlorosomes of Chloroflexus aurantiacus and B800-B850 antenna complexes of Rhodobacter sphaeroides

We have used a new and relatively easy approach to study the pigment-organization in chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus and in B800-850 antenna complexes of the photosynthetic purple bacterium Rhodobacter sphaeroides. These particles were embedded in compressed and uncompressed gels and the polarized fluorescence was determined in a 90° setup. Assuming both a rotational symmetric distribution of the particles in the gel and of the transition dipole moments in the particles, the order parameters <P₂> and <P₄>, describing the orientation of the symmetry axis of the particles with respect to the direction of gel expansion can be determined. Moreover, the direction parameters, describing the orientation of the absorption and emission dipole moments with respect to the symmetry axis of the particles can be obtained.

The value of <P₂> is essential for quantitative interpretation of linear dichroism measurements and usually it is estimated from theoretical approaches, which may lead to incorrect results. For the rod-like chlorosomes the value of <P₂> appears to be the same as predicted by the theoretical approach of Ganago, A. O., M. V. Fok, I. A. Abdourakhmanov, A. A. Solov'ev, and Yu. E. Erokhin (1980. Mol. Biol. [Mosc.]. 14:381-389). The agreement with linear dichroism results, analyzed with this theoretical approach shows that the transition dipole moments are indeed in good approximation distributed in a rotationally symmetric way around the long axis of the chlorosomes. Moreover, it appears those BChl c molecules, which fluoresce, are oriented in the same way with respect to the symmetry axis as the rest of these pigments, with the dipole moments close to parallel to the long axis.

The B800-850 complexes appear to orient like discs, whereas the transition dipoles of the BChl a 800- and 850-nm bands are oriented almost perpendicular to the symmetry axis. These findings are in agreement with the minimal model for these complexes proposed by Kramer, H. J. M., R. van Grondelle, C. N. Hunter, W. H. J. Westerhuis, and J. Amesz (1984. Biochim. Biophys. Acta. 156-165).

The amount of orientation of the particles appears to vary for different gels and it is lower than predicted by the theory of Ganago et al., showing that application of their approach for these particles leads to incorrect interpretations.

The approach that is used in this study allows determination of orientations of those dipole moments, which transfer their excitation energy to the fluorescing species, in contrast to linear dichroism measurements, where the orientations of all absorbing dipole moments are studied. For the polarized fluorescence measurements, the amount of orientation of the particles is determined experimentally, whereas for linear dichroism this amount has to be estimated from theoretical models. The value of <P₂> that can be determined from the fluorescence measurements can, however, also be used for a quantitative interpretation of the linear dichroism results.

     

Recent Emergence of Clonal Group O25b:K1:H4-B2-ST131 ibeA Strains among Escherichia coli Poultry Isolates,Including CTX-M-9-Producing Strains,and Comparison with Clinical Human Isolates

To discern the possible spread of the Escherichia coli O25b:H4-ST131 clonal group in poultry and the zoonotic potential of avian strains, we made a retrospective search of our strain collection and compared the findings for those strains with the findings for current strains. Thus, we have characterized a collection of 19 avian O25b:H4-ST131 E. coli strains isolated from 1995 to 2010 which, interestingly, harbored the ibeA gene. Using this virulence gene as a criterion for selection, we compared those 19 avian strains with 33 human O25b:H4-ST131 ibeA-positive E. coli strains obtained from patients with extraintestinal infections (1993 to 2009). All 52 O25b:H4-ST131 ibeA-positive E. coli strains shared the fimH, kpsMII, malX, and usp genes but showed statistically significant differences in nine virulence factors, namely, papGIII, cdtB, sat, and kpsMII K5, which were associated with human strains, and iroN, kpsMII K1, cvaC, iss, and tsh, which were associated with strains of avian origin. The XbaI macrorestriction profiles of the 52 E. coli O25b:H4-ST131 ibeA-positive strains revealed 11 clusters (clusters I to XI) of >85% similarity, with four clusters including strains of human and avian origin. Cluster VII (90.9% similarity) grouped 10 strains (7 avian and 3 human strains) that mostly produced CTX-M-9 and that also shared the same virulence profile. Finally, we compared the macrorestriction profiles of the 12 CTX-M-9-producing O25b:H4-ST131 ibeA strains (7 avian and 5 human strains) identified among the 52 strains with those of 15 human O25b:H4-ST131 CTX-M-14-, CTX-M-15-, and CTX-M-32-producing strains that proved to be negative for ibeA and showed that they clearly differed in the level of similarity from the CTX-M-9-producing strains. In conclusion, E. coli clonal group O25b:H4-ST131 ibeA has recently emerged among avian isolates with the new acquisition of the K1 capsule antigen and includes CTX-M-9-producing strains. This clonal group represents a real zoonotic risk that has crossed the barrier between human and avian hosts.Strains of the extensively antimicrobial-resistant Escherichia coli clonal group of sequence type (ST) 131 (ST131) belonging to serotype O25b:H4 have recently been recognized to be important human pathogens worldwide (9, 33). Although it is commonly associated with the dissemination of CTX-M-15 extended-spectrum cephalosporin resistance, E. coli O25b:H4-ST131 also occurs as a fluoroquinolone (FQ)-resistant but cephalosporin-susceptible pathogen (5, 22, 26, 27). Currently, it is assumed that O25b:H4-ST131 strains circulate not only among humans but also among animal hosts (13, 21, 37), which would contribute to the ongoing global emergence of O25b:H4-ST131, in the case of regular transmission between animals and humans. Even though CTX-M-15 is the most widely distributed extended-spectrum beta-lactamase (ESBL) linked to this clonal group, other, different variants of CTX-M have recently been reported, such as CTX-M-9, CTX-M-14, and CTX-M-32 (4, 34, 36, 39). Noteworthy was the detection, for the first time on poultry farms, of this clonal group producing CTX-M-9 that had macrorestriction profiles and virulence genes very similar to those observed in clinical human isolates (10).Extraintestinal pathogenic E. coli (ExPEC) strains, which include avian pathogenic E. coli (APEC) and human uropathogenic E. coli (UPEC), septicemic E. coli, and newborn meningitis-causing E. coli (NMEC) strains, exhibit considerable genome diversity and have a wide range of virulence-associated factors (12, 18). While infections caused by APEC strains initially start as a respiratory tract disease which evolves to a systemic infection of the internal organs and, finally, to sepsis, the most frequent origin of human sepsis is urinary tract infection (UTI), especially pyelonephritis (2, 3, 11). However, APEC strains have been recognized to share common traits with human isolates (29, 30, 31), including the K1 capsule antigen (23, 24, 29) and the ibeA gene (14). In addition, retail chicken products have been found to carry nalidixic-resistant ExPEC strains (17, 19), and although it is drug susceptible, an E. coli strain belonging to the O25b:H4-ST131 clonal group has even recently been detected in retail chicken (41), supporting the urgent necessity for the implementation of food control measures.The aim of the present study was to discern the possible spread of the O25b:H4-ST131 clonal group, especially CTX-M-9-producing strains, in poultry and the zoonotic potential of avian isolates. For this purpose, we made a retrospective search of our human and avian strain collections and compared the findings for those strains with the findings for current strains. Identification of this emerging clone among avian sources and comparison of the clone with clinical human isolates will shed new light on the epidemiology of the O25b:H4-ST131 clonal group.     

Association of CHRNA5-A3-B4 Variation with Esophageal Squamous Cell Carcinoma Risk and Smoking Behaviors in a Chinese Population

Background

CHRNA5-A3-B4, the gene cluster encoding nicotinic acetylcholine receptor subunits, is associated with lung cancer risk and smoking behaviors in people of European descent. Because cigarette smoking is also a major risk factor for esophageal squamous cell carcinoma (ESCC), we investigated the associations between variants in CHRNA5-A3-B4 and ESCC risk, as well as smoking behaviors, in a Chinese population.

Methods

A case-control study of 866 ESCC patients and 952 healthy controls was performed to study the association of polymorphisms (rs667282 and rs3743073) in CHRNA5-A3-B4 with cancer risk using logistic regression models. The relationships between CHRNA5-A3-B4 polymorphisms and smoking behaviors that can be quantified by cigarettes smoked per day (CPD) and pack-years of smoking were separately estimated with Kruskal-Wallis tests among all 840 smokers.

Results

CHRNA5-A3-B4 rs667282 TT/TG genotypes were associated with significantly increased risk of ESCC [adjusted odds ratio (OR) = 1.32, 95% confidence interval (CI) = 1.03 – 1.69, P = 0.029]. The increased ESCC risk was even higher among younger subjects (≤60 years) (OR = 1.44, 95% CI = 1.04 – 1.98, P = 0.024). These effects were not found in another polymorphism rs3743073. No evident association between the two polymorphisms and smoking behaviors was observed.

Conclusions

These results support the hypothesis that CHRNA5-A3-B4 is a susceptibility gene cluster for ESCC. The relationship between CHRNA5-A3-B4 and smoking behaviors in a Chinese population needs further investigation.     

Structure and absolute stereochemistry of tomenphantopin-A and -B two cytotoxic sesquiterpene lactones from elephantopus tomentosus

The structure and absolute stereochemistry of the new cytotoxic sesquiterpene lactones, tomenphantopin-A and -B, isolated from Elephantopus tomentosus, were determined from spectral data, chemical transformation and single-crystal X-ray analysis. Tomenphantopin-A and -B are two elephantopin-type germacranolides which possess an α-orientation of the oxygen substituent at C-2.     

Protective human leucocyte antigen haplotype, HLA-DRB1*01-B*14, against chronic Chagas disease in Bolivia

Background

Chagas disease, caused by the flagellate parasite Trypanosoma cruzi affects 8–10 million people in Latin America. The mechanisms that underlie the development of complications of chronic Chagas disease, characterized primarily by pathology of the heart and digestive system, are not currently understood. To identify possible host genetic factors that may influence the clinical course of Chagas disease, Human Leucocyte Antigen (HLA) regional gene polymorphism was analyzed in patients presenting with differing clinical symptoms.

Methodology

Two hundred and twenty nine chronic Chagas disease patients in Santa Cruz, Bolivia, were examined by serological tests, electrocardiogram (ECG), and Barium enema colon X-ray. 31.4% of the examinees showed ECG alterations, 15.7% megacolon and 58.1% showed neither of them. A further 62 seropositive megacolon patients who had undergone colonectomy due to acute abdomen were recruited. We analyzed their HLA genetic polymorphisms (HLA-A, HLA-B, MICA, MICB, DRB1 and TNF-alpha promoter region) mainly through Sequence based and LABType SSO typing test using LUMINEX Technology.

Principal Findings

The frequencies of HLA-DRB1*01 and HLA-B*14:02 were significantly lower in patients suffering from megacolon as well as in those with ECG alteration and/or megacolon compared with a group of patients with indeterminate symptoms. The DRB1*0102, B*1402 and MICA*011 alleles were in strong Linkage Disequilibrium (LD), and the HLA-DRB1*01-B*14-MICA*011haplotype was associated with resistance against chronic Chagas disease.

Conclusions

This is the first report of HLA haplotype association with resistance to chronic Chagas disease.     

Stratification by Smoking Status Reveals an Association of CHRNA5-A3-B4 Genotype with Body Mass Index in Never Smokers

We previously used a single nucleotide polymorphism (SNP) in the CHRNA5-A3-B4 gene cluster associated with heaviness of smoking within smokers to confirm the causal effect of smoking in reducing body mass index (BMI) in a Mendelian randomisation analysis. While seeking to extend these findings in a larger sample we found that this SNP is associated with 0.74% lower body mass index (BMI) per minor allele in current smokers (95% CI -0.97 to -0.51, P = 2.00×10⁻¹⁰), but also unexpectedly found that it was associated with 0.35% higher BMI in never smokers (95% CI +0.18 to +0.52, P = 6.38×10⁻⁵). An interaction test confirmed that these estimates differed from each other (P = 4.95×10⁻¹³). This difference in effects suggests the variant influences BMI both via pathways unrelated to smoking, and via the weight-reducing effects of smoking. It would therefore be essentially undetectable in an unstratified genome-wide association study of BMI, given the opposite association with BMI in never and current smokers. This demonstrates that novel associations may be obscured by hidden population sub-structure. Stratification on well-characterized environmental factors known to impact on health outcomes may therefore reveal novel genetic associations.     

Silencing of CD44 Gene Expression in Human 143-B Osteosarcoma Cells Promotes Metastasis of Intratibial Tumors in SCID Mice

Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells'' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.     

Geometry, thermodynamics, and protein

We derive a new continuous free energy formula for protein folding. We obtain the formula first by adding hydrophobic effect to a classical free energy formula for cavities in water. We then obtain the same formula by geometrically pursuing the structure that fits best the well-known global geometric features of native structures of globular proteins: 1. high density; 2. small surface area; 3. hydrophobic core; 4. forming domains for long polypeptide chains. Conformations of a protein are presented as an all atom CPK model where each atom is a ball B(x_i,r_i). All conformations satisfy generally defined steric conditions. For each conformation P of a globular protein, there is a closed thermodynamic system Ω_P⊃P bounded by the molecular surface M_P. Both methods derive the same free energy aV(P)+bA(P)+cW(P), where a,b,c>0, V(P), A(P), and W(P) are volume of Ω_P, area of M_P, and area of the hydrophobic surface W_P⊂M_P, which quantifies hydrophobic effect.Minimizing W(P) is sufficient to produce statistically significant native like secondary structures and hydrogen bonds in the proteins we simulated.     

Isolation and characterization of the plant immune-priming compounds Imprimatin B3 and -B4, potentiators of disease resistance in Arabidopsis thaliana

Plant activators are chemical crop protectants that fortify the immune system in plants. Unlike pesticides that target pathogens, plant activators provide durable effects against a broad spectrum of diseases, which have not been overcome by pathogenic microbes. Plant activators are not only useful agrochemicals, but can also help to elucidate the details of the plant immune system. Using an established high-throughput screening procedure, we previously identified 5 compounds, designated as Imprimatins, which prime plant immune response. These compounds increased disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants by inhibiting 2 salicylic acid (SA) glucosyltransferases (SAGTs), resulting in accumulation of the phytohormone SA. Here, we report the isolation of 2 additional Imprimatins, B3 and B4, which are structurally similar to Imprimatin B1 and B2. Because these compounds did not have strong inhibitory effects on SAGTs in vitro, they may exert their function after metabolic conversion in vivo.