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1.  Induction of KLF4 in response to heat stress 
Cell Stress & Chaperones  2006;11(4):379-389.
Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger–containing transcription factor with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. However, little is known about the response of KLF4 to heat stress. In this study, Western blot and reverse transcriptase–polymerase chain reaction were performed to determine the changes in KLF4 expression in response to heat stress. The results showed that heat stress up-regulated KLF4 messenger RNA and protein levels in a time-dependent manner in vivo and in 4 cell lines. Moreover, a study with heat shock transcription factor 1 (Hsf1) gene knockout mice indicated that the induction of KLF4 in response to heat stress was mediated by Hsf1. This process occurred rapidly, indicating that KLF4 is an immediate early response gene of heat stress. Next, the roles of KLF4 under heat stress conditions were analyzed for cells overexpressing or deficient in KLF4. The results showed overexpression of KLF4 increased the death rate of C2C12 cells, whereas KLF4 deficiency decreased the injury of C2C12 cells from heat stress conditions, suggesting that KLF4 might play an important role in cell injury induced by heat stress. KLF4 might be an immediate early response gene and could play an important role in cell injury induced by heat stress.
doi:10.1379/CSC-210.1
PMCID: PMC1712684  PMID: 17278886
2.  Development of a Serotype-Specific DNA Microarray for Identification of Some Shigella and Pathogenic Escherichia coli Strains▿  
Journal of Clinical Microbiology  2006;44(12):4376-4383.
Shigella and pathogenic Escherichia coli are major causes of human infectious diseases and are responsible for millions of cases of diarrhea worldwide every year. A convenient and rapid method to identify highly pathogenic serotypes of Shigella and E. coli is needed for large-scale epidemiologic study, timely clinical diagnosis, and reliable quarantine of the pathogens. In this study, a DNA microarray targeting O-serotype-specific genes was developed to detect 15 serotypes of Shigella and E. coli, including Shigella sonnei; Shigella flexneri type 2a; Shigella boydii types 7, 9, 13, 16, and 18; Shigella dysenteriae types 4, 8, and 10; and E. coli O55, O111, O114, O128, and O157. The microarray was tested against 186 representative strains of all Shigella and E. coli O serotypes, 38 clinical isolates, and 9 strains of other bacterial species that are commonly present in stool samples and was shown to be specific and reproducible. The detection sensitivity was 50 ng genomic DNA or 104 CFU per ml in mock stool specimens. This is the first report of a microarray for serotyping Shigella and pathogenic E. coli. The method has a number of advantages over traditional bacterial culture and antiserum agglutination methods and is promising for applications in basic microbiological research, clinical diagnosis, food safety, and epidemiological surveillance.
doi:10.1128/JCM.01389-06
PMCID: PMC1698391  PMID: 17021058
3.  Genetic Polymorphisms of Phase II Metabolic Enzymes and Lung Cancer Susceptibility in a Population of Central South China 
Disease Markers  2006;22(3):141-152.
A case-control study was conducted for analyzing the genetic polymorphisms of phase II metabolic enzymes in 97 patients with lung cancer and 197 healthy subjects from Han ethnic group of Hunan Province located in Central South China. The results showed that the frequencies of glutathione S-transferase (GST) M1-null (GSTM1-) or GSTT1-null (GSTT1-) genotype alone, or combined form of both in lung cancer patients were significantly higher than those of the controls. Genotypes of combining GSTP1 mutant/GSTM1(-) or GSTP1 mutant/GSTT1(-) led to high risk of lung cancer. Individuals carrying any two or all three of GSTM1(-), GSTT1(-) and GSTP1 mutant genotypes have a distinctly increased risk of lung cancer when compared to those with GSTM1 present (GSTM1+: GSTM1+/+ or GSTM1+/−), GSTT1 present (GSTT1+: GSTT1+/+ or GSTT1+/−) and GSTP1 wild genotypes. Furthermore, individuals possessing combined genotypes of N-acetyltransferase 2 (NAT2) rapid acetylator, GSTP1 mutant and both GSTT1(-) and GSTM1(-) have a remarkably higher lung cancer risk than those carrying combined NAT2 slow acetylator genotype, GSTP1 wild genotype and both GSTT1(+) and GSTM1(+) genotypes. All these findings suggest that the genetic polymorphisms of phase II metabolic enzymes affect the susceptibility of lung cancer in the Han ethnic group of Central South China.
doi:10.1155/2006/436497
PMCID: PMC3851385  PMID: 16788248
Genetic polymorphism; GST; NAT2; lung cancer; susceptibility
4.  Human Streptococcus suis Outbreak, Sichuan, China 
Emerging Infectious Diseases  2006;12(6):914-920.
Streptococcus suis outbreak was associated with exposure to sick or dead pigs.
From mid-July to the end of August 2005, a total of 215 cases of human Streptococcus suis infections, 66 of which were laboratory confirmed, were reported in Sichuan, China. All infections occurred in backyard farmers who were directly exposed to infection during the slaughtering process of pigs that had died of unknown causes or been killed for food because they were ill. Sixty-one (28%) of the farmers had streptococcal toxic shock syndrome; 38 (62%) of them died. The other illnesses reported were sepsis (24%) and meningitis (48%) or both. All isolates tested positive for genes for tuf, species-specific 16S rRNA, cps2J, mrp, ef, and sly. A single strain of S. suis caused the outbreak, as shown by the identification of a single ribotype. The high death ratio was of concern; prohibiting backyard slaughtering ended the outbreak.
doi:10.3201/eid1206.051194
PMCID: PMC3373052  PMID: 16707046
Keywords: Streptococcus suis; streptococcal toxic shock syndrome; outbreak; zoonoses; research
5.  Identification and developmental expression analysis of a novel homeobox gene closely linked to the mouse Twirler mutation 
Gene expression patterns : GEP  2006;6(6):10.1016/j.modgep.2005.11.012.
The Twirler mutation arose spontaneously and causes inner ear defects in heterozygous and cleft lip and/or cleft palate in homozygous mutant mice, providing a unique animal model for investigating the molecular mechanisms of inner ear and craniofacial development. Here we report the identification of a novel homeobox gene, Iroquois-related homeobox like-1 (Irxl1), from the Twirler locus. Irxl1 encodes a TALE-family homeodomain protein with its homeodomain exhibiting the highest amino acid sequence identity (54%) to those of invertebrate Iroquois and vertebrate Irx subfamily members. The putative Irxl1 protein lacks the Iro-box, a conserved motif in all known members of the Irx subfamily. Searching the databases showed that Irxl1 orthologs exist in Xenopus, chick, and mammals. In situ hybridization analyses of mouse embryos at various developmental stages showed that Irxl1 mRNA is highly expressed in the frontonasal process and palatal mesenchyme during primary and secondary palate development. In addition, Irxl1 mRNA is strongly expressed in mesenchyme surrounding the developing inner ear, in discrete regions of the developing mandible, in the dermamyotome during somite differentiation, and in a subset of muscular structures in late embryonic stages. The developmental expression pattern indicates that Irxl1 is a good candidate gene for the Twirler gene.
doi:10.1016/j.modgep.2005.11.012
PMCID: PMC3869088  PMID: 16426902
cleft lip; cleft palate; craniofacial development; dermamyotome; homeobox; homeodomain; Hox; intervertebral disc; Iroquois; Irx; Irxl1; mandible; palate development; secondary palate; skeletal muscle; somite; TALE; Twirler mutation
6.  Immersed finite element method and its applications to biological systems 
This paper summarizes the newly developed immersed finite element method (IFEM) and its applications to the modeling of biological systems. This work was inspired by the pioneering work of Professor T.J.R. Hughes in solving fluid–structure interaction problems. In IFEM, a Lagrangian solid mesh moves on top of a background Eulerian fluid mesh which spans the entire computational domain. Hence, mesh generation is greatly simplified. Moreover, both fluid and solid domains are modeled with the finite element method and the continuity between the fluid and solid subdomains is enforced via the interpolation of the velocities and the distribution of the forces with the reproducing Kernel particle method (RKPM) delta function. The proposed method is used to study the fluid–structure interaction problems encountered in human cardiovascular systems. Currently, the heart modeling is being constructed and the deployment process of an angioplasty stent has been simulated. Some preliminary results on monocyte and platelet deposition are presented. Blood rheology, in particular, the shear-rate dependent de-aggregation of red blood cell (RBC) clusters and the transport of deformable cells, are modeled. Furthermore, IFEM is combined with electrokinetics to study the mechanisms of nano/bio filament assembly for the understanding of cell motility.
doi:10.1016/j.cma.2005.05.049
PMCID: PMC2830735  PMID: 20200602
Immersed finite element method; Reproducing Kernel particle method; Fluid–structure interaction; Cytoskeletal dynamics; Red blood cell; Aggregation; Thrombosis; Cardiovascular system; Micro-circulation; Nano-electro-mechanical-sensors; Cell motility; Surgical corrective procedures
7.  A genomewide scan for quantitative trait loci underlying areal bone size variation in 451 Caucasian families 
Journal of Medical Genetics  2006;43(11):873-880.
Background
Bone size is an important determinant of bone strength and is under strong genetic control.
Objective
To identify quantitative trait loci (QTL) for areal bone size variation, a large‐scale genomewide linkage scan was carried out in 451 Caucasian families.
Participants and methods
Of 4124 people with phenotypes, 3899 were genotyped with 410 microsatellite markers. Multipoint linkage analyses were carried out in the entire sample, as well as in men and women separately. Potential epistatic interactions between identified genomic regions were also assessed.
Results
Several potentially important genomic regions were identified, such as 8q24 for hip bone size (logarithm of the ratio of the odds that two loci are linked (LOD) 3.27) and 2p24 (LOD 2.04) for spine bone size. 8q24 may also interact with 19p13 to affect hip bone size. Several sex‐specific QTL were also detected, such as 14q21 (LOD 2.94) for wrist bone size in women and 16q12 (LOD 2.19) for hip bone size in men.
Conclusions
Together with previous findings, this study has further delineated the genetic basis of bone size and laid a foundation for future studies to eventually elucidate the mechanisms of bone size regulation and associated fracture risks.
doi:10.1136/jmg.2006.041251
PMCID: PMC2563191  PMID: 16825438
8.  Expression of cadherin10, a type II classic cadherin gene, in the nervous system of the embryonic zebrafish 
Gene expression patterns : GEP  2006;6(7):703-710.
Cadherins are cell surface adhesion molecules that play important roles in development of tissues and organs. In this study we analyzed expression pattern of cadherin10, a member of the type II classic cadherin subfamily, in the embryonic zebrafish using in situ hybridization methods. cadherin10 message (cdh10) is first and transiently expressed by the notochord. In the developing nervous system, cdh10 was first detected in a subset of the cranial ganglia, then in restricted brain regions and neural retina. As development proceeds, cdh10 expression domain and/or expression levels increased in the embryonic nervous system. Our results show that cdh10 expression in the zebrafish developing nervous system is both spatially and temporally regulated.
doi:10.1016/j.modgep.2005.12.009
PMCID: PMC2562320  PMID: 16488669
zebrafish; development; cell adhesion molecules; brain; cranial and lateral line ganglia
9.  The role of protein dynamics in thymidylate synthase catalysis 
Biochemistry  2006;45(24):7415-7428.
The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2′-deoxyuridine 5′-monophosphate (dUMP) to 2′-deoxythymidine 5′-monophosphate. Using kinetic and x-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3′-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Kms for both substrate and the cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of E. coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.
doi:10.1021/bi060152s
PMCID: PMC2556892  PMID: 16768437
10.  Ebselen Induced C6 Glioma Cell Death in Oxygen and Glucose Deprivation 
Chemical research in toxicology  2006;19(5):655-660.
Studies have shown that ebselen is an anti-inflammatory and antioxidative agent. Its protective effect has been investigated in oxidative stress related diseases such as cerebral ischemia in recent years. However, experimental evidence also shows that ebselen causes cell death in several different cell types. Whether ebselen will have beneficial or detrimental effect on cells under ischemic condition is not known. Herein, we studied the effect of ebselen on C6 glioma cell under oxygen and glucose deprivation (OGD), an in vitro ischemic model. We found that ebselen significantly enhanced cell death after three-hour OGD as observed by lactase dehydrogenase (LDH) release and cellular morphological changes. Further studies revealed that depletion of cellular glutathione level by the combined action of ebselen and OGD played a role in enhanced cell death as demonstrated by the following evidence: 1) Cellular GSH was significantly depleted by combined effort of ebselen and OGD, compared to ebselen or OGD insult alone; 2) Exogenous addition of N-acetyl cysteine completely diminished the cell damage induced by ebselen and OGD; 3) Supplement of glucose, which provides cellular reducing agents and thus maintains cellular GSH level, to the OGD medium diminished C6 cell damage induced by ebselen. We conclude that depleting cellular glutathione plays an important role in ebselen-induced cell death with OGD. Our results suggest that ebselen can have beneficial or toxic effect, depending on the availability of GSH.
doi:10.1021/tx0502544
PMCID: PMC2556889  PMID: 16696567
11.  Biosynthesis of Fosfomycin, Re-examination and Re-confirmation of A Unique Fe(II) and NAD(P)H-dependent Epoxidation Reaction 
Biochemistry  2006;45(38):11473-11481.
(S)-2-hydroxypropylphosphonic acid epoxidase (HppE) catalyzes the epoxide ring closure of (S)-HPP to form fosfomycin, a clinically useful antibiotic. Early investigation showed that its activity can be reconstituted with Fe(II), FMN, NADH, and O2, and identified HppE as a new type of mononuclear non-heme iron-dependent oxygenase involving high valent iron-oxo species in the catalysis. However, a recent study showed that the Zn(II)-reconstituted HppE is active, and HppE exhibits modest affinity for FMN. Thus, a new mechanism is proposed in which the active site bound Fe2+ or Zn2+ serves as a Lewis acid to activate the 2-OH group of (S)-HPP, and the epoxide ring is formed by the attack of the 2-OH group at C-1 coupled with the transfer of the C-1 hydrogen as a hydride ion to the bound FMN. To distinguish between these mechanistic discrepancies, we re-examined the bioautography assay, the basis for the alternative mechanism, and showed that Zn(II) cannot replace Fe(II) in the HppE reaction, and NADH is indispensable. Moreover, we demonstrated that the proposed role for FMN as a hydride acceptor is inconsistent with the finding that FMN cannot bind to HppE in the presence of substrate. In addition, using a newly developed HPLC assay we showed that several non-flavin electron mediators could replace FMN in the HppE-catalyzed epoxidation. Taken together, these results argue against the newly proposed “nucleophilic displacement-hydride transfer” mechanism, but are fully consistent with the previously proposed iron-redox mechanism for HppE catalysis, which is unique within the mononuclear non-heme iron enzyme superfamily.
doi:10.1021/bi060839c
PMCID: PMC2515266  PMID: 16981707
13.  Differential Expression of Photoreceptor-Specific Genes in the Retina of a Zebrafish Cadherin2 Mutant glass onion and Zebrafish Cadherin4 Morphants 
Experimental eye research  2006;84(1):163-175.
Cadherins are Ca++-dependent transmembrane molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin2 (also called N-cadherin) and cadherin4 (also called R-cadherin), members of the classic cadherin subfamily, have been shown to be involved in development of a variety of tissues and organs including the visual system. To gain insight into cadherin2 and cadherin4 function in differentiation of zebrafish photoreceptors, we have analyzed expression patterns of several photoreceptor-specific genes (crx, gnat1, gnat2, irbp, otx5, rod opsin, rx1, and uv opsin) and/or a cone photoreceptor marker (zpr-1) in the retina of a zebrafish cadherin2 mutant, glass onion (glo) and in zebrafish embryos injected with a cadherin4 specific antisense morpholino oligonucleotide (cdh4MO). We find that expression of all these genes, and of zpr-1, is greatly reduced in the retina of both the glo and cadherin4 morphants. Moreover, in these embryos, expression of some genes (e.g. gnat1, gnat2 and irbp) is more affected than others (e.g.rod opsin and uv opsin). In embryos with both cadherins functions blocked (glo embryos injected with the cdh4MO), the eye initially formed, but became severely and progressively disintegrated and expressed little or no crx and otx5 as development proceeded. Our results suggest that cadherin2 and cadherin4 play important roles in the differentiation of zebrafish retinal photoreceptors.
doi:10.1016/j.exer.2006.09.011
PMCID: PMC1853368  PMID: 17070801
retina; rods and cones; development; cell adhesion molecules; Danio rerio
14.  Cloning and expression analysis of cadherin7 in the central nervous system of the embryonic zebrafish 
Gene expression patterns : GEP  2006;7(1-2):15-22.
Cadherin cell adhesion molecules exhibit unique expression patterns during development of the vertebrate central nervous system. In this study we obtained a full-length cDNA of a novel zebrafish cadherin using reverse transcriptase-polymerase chain reaction (RT-PCR) and 5′ and 3′ rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of this molecule is most similar to the published amino acid sequences of chicken and mammalian cadherin7 (Cdh7), a member of the type II cadherin subfamily. cadherin7 message (cdh7) expression in embryonic zebrafish was studied using in situ hybridization and RT-PCR methods. cdh7 expression begins at about 12 hours post fertilization (hpf) in a small patch in the anterior neural keel, and along the midline of the posterior neural keel. By 24 hpf, cdh7 expression in the brain shows a distinct segmental pattern that reflects the neuromeric organization of the brain, while its expression domain in the spinal cord is continuous, but confined to the middle region of the spinal cord. As development proceeds, cdh7 expression is detected in more regions of the brain, including the major visual structures in the fore- and midbrains, while its expression domain in the hindbrain becomes more restricted, and its expression in the spinal cord becomes undetectable. cdh7 expression becomes reduced in 3-day old embryos. Our results show that cdh7 expression in the zebrafish developing central nervous system is both spatially and temporally regulated.
doi:10.1016/j.modgep.2006.05.002
PMCID: PMC1716651  PMID: 16774849
zebrafish; development; cell adhesion molecules; brain; spinal cord; visual system
15.  Successful Radiotherapy of Tumor in Pretargeted Mice by 188Re-Radiolabeled Phosphorodiamidate Morpholino Oligomer, a Synthetic DNA Analogue 
Purpose
Pretargeting has been attracting increasing attention as a drug delivery approach. We recently proposed Watson-Crick pairing of phosphorodiamidate morpholino oligomers (MORF) for the recognition system in tumor pretargeting. MORF pretargeting involves the initial i.v. injection of a MORF-conjugated antitumor antibody and the subsequent i.v. injection of the radiolabeled complement. Our laboratory has reported on MORF pretargeting for diagnosis using 99mTc as radiolabel. We now report on the use of MORF pretargeting for radiotherapy in a mouse tumor model using 188Re as the therapeutic radiolabel.
Experimental Design
An initial tracer study was done to estimate radiation dose, and was followed by the radiotherapy study at 400 μCi per mouse with three control groups (untreated, MORF antibody alone, and 188Re complementary MORF alone).
Results
Tracer study indicated rapid tumor localization of 188Re and rapid clearance from normal tissues with a tumor area under the curve (AUC) about four times that of kidney and blood (the normal organs with highest radioactivity). Tumor growth in the study group ceased 1 day after radioactivity injection, whereas tumors continued to grow at the same rate among the three control groups. At sacrifice on day 5, the average net tumor weight in the study group was significantly lower at 0.68 ± 0.29 g compared with the three control groups (1 24 ± 0.31g, 1 25 ± 0.39 g, and 1 35 ± 0.41g; Ps <0.05), confirming the therapeutic benefit observed by tumor size measurement.
Conclusions
MORF pretargeting has now been shown to be a promising approach for tumor radiotherapy as well as diagnosis.
doi:10.1158/1078-0432.CCR-06-0844
PMCID: PMC1587615  PMID: 16914585
16.  cadherin-6 message Expression in the Nervous System of Developing Zebrafish 
Cadherins are cell surface adhesion molecules that play important roles in development of a variety of tissues including the nervous system. In this study we analyzed expression pattern of cadherin-6, a member of the type II cadherin subfamily, in the embryonic zebrafish nervous system using in situ hybridization methods. cadherin-6 message is first expressed by the neural keel, then by restricted regions in the brain and spinal cord. cadherin-6 expression in the brain transiently delineates specific brain regions. In the peripheral nervous system, cadherin-6 message is expressed by the neurogenic placodes and the dorsal root ganglia. As development proceeds, cadherin-6 expression domain and/or expression levels increased in the embryonic nervous system. Our results show that cadherin-6 expression in the zebrafish developing nervous system is both spatially and temporally regulated, implicating a role for cadherin-6 in the formation of these nervous structures.
doi:10.1002/dvdy.20607
PMCID: PMC1360229  PMID: 16258934
zebrafish; development; cell adhesion molecules; brain, cranial ganglia, lateral line system
17.  Familial Aggregation in Skin Flush Response to Niacin Patch Among Schizophrenic Patients and Their Nonpsychotic Relatives 
Schizophrenia Bulletin  2006;33(1):174-182.
Though a reduced flush response to niacin has been found in schizophrenic patients, whether it is a vulnerability indicator to schizophrenia remains little known. We aimed to examine the familial aggregation in niacin flush response among schizophrenic patients and their nonpsychotic relatives. In a sample of 153 schizophrenia probands, 217 parents, 70 siblings, and 94 normal subjects, 3 concentrations (0.001 M, 0.01 M, and 0.1 M) of niacin were applied to the forearm skin and the flush response was rated at 5, 10, and 15 minutes, respectively, with a 4-point scale. Both the heritability for continuous flush scores and the recurrence risk ratios for binary nonflush response in the nonpsychotic relatives of schizophrenic patients were estimated, and ordinal logistic regression analyses of relatives' niacin response on probands' were further conducted to adjust for potential confounders. The greatest heritabilities ranged from 47% (0.01 M at 10 minutes) to 54% (0.1 M at 5 minutes). The risk ratios of 0.01 M at 10 minutes (ranging from 2.60 for using score 1 or less to 5.06 for using score 0 as nonflush) and 5 minutes (1.66 for using score 0 as nonflush) were significantly greater than one. Multiple ordinal logistic regression analyses further revealed that the association between probands and relatives in niacin flush response remained after adjustment for potential confounders, including age, sex, allergy, tobacco smoking, and coffee drinking. These findings provide support for the potential of niacin flush response as a vulnerability indicator to schizophrenia.
doi:10.1093/schbul/sbl038
PMCID: PMC2632299  PMID: 16936284
nicotinic acid; prostaglandin; heritability; recurrence risk ratio; vulnerability indicator
18.  A Novel αB-Crystallin Mutation Associated with Autosomal Dominant Congenital Lamellar Cataract 
Purpose
To identify the mutation and the underlying mechanism of cataractogenesis in a five-generation autosomal dominant congenital lamellar cataract family.
Methods
Nineteen mutation hot spots associated with autosomal dominant congenital cataract have been screened by PCR-based DNA sequencing. Recombinant wild-type and mutant human αB-crystallin were expressed in Escherichia coli and purified to homogeneity. The recombinant proteins were characterized by far UV circular dichroism, intrinsic tryptophan fluorescence, Bis-ANS fluorescence, multiangle light-scattering, and the measurement of chaperone activity.
Results
A novel missense mutation in the third exon of the αB-crystallin gene (CRYAB) was found to cosegregate with the disease phenotype in a five-generation autosomal dominant congenital lamellar cataract family. The single-base substitution (G→A) results in the replacement of the aspartic acid residue by asparagine at codon 140. Far UV circular dichroism spectra indicated that the mutation did not significantly alter the secondary structure. However, intrinsic tryptophan fluorescence spectra and Bis-ANS fluorescence spectra indicated that the mutation resulted in alterations in tertiary and/or quaternary structures and surface hydrophobicity of αB-crystallin. Multiangle light-scattering measurement showed that the mutant αB-crystallin tended to aggregate into a larger complex than did the wild-type. The mutant αB-crystallin was more susceptible than wild-type to thermal denaturation. Furthermore, the mutant αB-crystallin not only lost its chaperone-like activity, it also behaved as a dominant negative which inhibited the chaperone-like activity of wild-type αB-crystallin.
Conclusions
These data indicate that the altered tertiary and/or quaternary structures and the dominant negative effect of D140N mutant αB-crystallin underlie the molecular mechanism of cataractogenesis of this pedigree.
doi:10.1167/iovs.05-1004
PMCID: PMC2078606  PMID: 16505043
19.  IL-18 stimulates IL-13-mediated IFN-γ-sensitive host resistance in vivo 
European journal of immunology  2006;36(5):1187-1198.
IL-4 and IL-13 are up-regulated during in vivo responses to many nematode parasites, but increasing evidence suggests that increases in IL-13 can also occur independently of the IL-4-dominant Th2 response. Blocking B7 after Trichuris muris inoculation inhibits resistance and IL-4 elevations, instead resulting in an IFN-γ-dominant response associated with susceptibility. However, blocking IFN-γ under these conditions restores IL-13-dependent resistance. In this study, we examined the mechanism of IL-13 up-regulation and associated protection during this in vivo immune response. CD4+ T cells and DX5+TCR− cells were identified as the major producers of IL-13, and the DX5+TCR− cells were phenotyped as NK cells, since they expressed CD11b, IL-2Rβ and Ly49C but not c-kit or FcεRI. NK cell-derived IL-13 elevations were Tcell-dependent, as CD4+ Tcell depletion blocked IL-13 production by mesenteric lymph node cells and induced susceptibility. IL-13 expression was increased independently of IL-12; however, blocking IL-18 function inhibited IL-13 production and increased susceptibility. These results indicate that CD4+ T cells and NK cells are the major sources of IL-13 during the in vivo Th1 response induced by B7 blockade and that under these conditions, IL-18 is specifically required for the in vivo up-regulation of IL-13 production and associated host protection.
doi:10.1002/eji.200535668
PMCID: PMC2000330  PMID: 16568498
Cytokines; Natural killer cells; Parasitichelminth; Rodent; T helper cells
20.  α-Synuclein Blocks ER-Golgi Traffic and Rab1 Rescues Neuron Loss in Parkinson’s Models 
Science (New York, N.Y.)  2006;313(5785):324-328.
Alpha-synuclein (αSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson’s disease (PD). In yeast cells and in neurons αSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following αSyn expression in yeast was a block in endoplasmic reticulum (ER)–to–Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic αSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against αSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.
doi:10.1126/science.1129462
PMCID: PMC1983366  PMID: 16794039
21.  Repeated 100 Hz TENS for the Treatment of Chronic Inflammatory Hyperalgesia and Suppression of Spinal Release of Substance P in Monoarthritic Rats 
Transcutaneous electrical nerve stimulation (TENS) has been shown to be an effective measure for pain relief. The aim of the present study was to determine the optimal intensity and interval of repeated 100 Hz TENS for the treatment of chronic inflammatory hyperalgesia in a monoarthritic pain model of the rat, and to assess the changes of the spinal substance P (SP) release in response to TENS treatment. A reliable, reproducible chronic monoarthritic pain model was produced by intra-articular injection of complete Freund's adjuvant (CFA) at single ankle joint. The efficacy of 100 Hz TENS treatments with different frequencies and intensities was compared. In the acute period (within 3 weeks) of monoarthritis, twice-a-week schedule of TENS reduced the swelling of the inflamed ankle significantly. In the stable period (4–9 weeks), however, once-a-week schedule produced a significantly better therapeutic effect on both inflammation and arthritic hyperalgesia than that of twice- or five-times-a-week schedule. Using three levels of intensity of TENS, we found that the weaker (1-1-2 mA) stimulation produced significantly better therapeutic effects. Repeated TENS produced a reduction of SP content in spinal perfusate in parallel with the progressive reduction of the arthritic pain scores. Our results suggest that (i) consecutive TENS treatments produced cumulative effect for chronic hyperalgesia, (ii) for chronic inflammatory hyperalgesia, a weaker intensity and more sparsely arranged treatment schedule may produce better therapeutic effect and (iii) a decrease in SP release may serve as one of the possible neurochemical mechanisms underlying the therapeutic effects of multiple TENS treatments on chronic inflammatory hyperalgesia.
doi:10.1093/ecam/nel056
PMCID: PMC1810365  PMID: 17342243
chronic inflammatory hyperalgesia; Freund's adjuvant; monoarthritis; substance P; transcutaneous electrical nerve stimulation (TENS)
22.  Engineered Vaginal Lactobacillus Strain for Mucosal Delivery of the Human Immunodeficiency Virus Inhibitor Cyanovirin-N 
Antimicrobial Agents and Chemotherapy  2006;50(10):3250-3259.
Women are at significant risk of human immunodeficiency virus (HIV) infection, with the cervicovaginal mucosa serving as a major portal for virus entry. Female-initiated preventatives, including topical microbicides, are urgently needed to help curtail the HIV/AIDS pandemic. Here we report on the development of a novel, live microbicide that employs a natural vaginal strain of Lactobacillus jensenii engineered to deliver the potent HIV inhibitor cyanovirin-N (CV-N). To facilitate efficient expression of CV-N by this bacterium, the L. jensenii 1153 genome was sequenced, allowing identification of native regulatory elements and sites for the chromosomal integration of heterologous genes. A CV-N expression cassette was optimized and shown to produce high levels of structurally intact CV-N when expressed in L. jensenii. Lactobacillus-derived CV-N was capable of inhibiting CCR5-tropic HIVBaL infectivity in vitro with a 50% inhibitory concentration of 0.3 nM. The CV-N expression cassette was stably integrated as a single copy into the bacterial chromosome and resolved from extraneous plasmid DNA without adversely affecting the bacterial phenotype. This bacterial strain was capable of colonizing the vagina and producing full-length CV-N when administered intravaginally to mice during estrus phase. The CV-N-producing Lactobacillus was genetically stable when propagated in vitro and in vivo. This work represents a major step towards the development of an inexpensive yet durable protein-based microbicide to block the heterosexual transmission of HIV in women.
doi:10.1128/AAC.00493-06
PMCID: PMC1610104  PMID: 17005802
24.  Mesenchymal Stem Cell-Mediated Functional Tooth Regeneration in Swine 
PLoS ONE  2006;1(1):e79.
Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.
doi:10.1371/journal.pone.0000079
PMCID: PMC1762318  PMID: 17183711
25.  Comprehensive analysis of the ATM, CHEK2 and ERBB2 genes in relation to breast tumour characteristics and survival: a population-based case-control and follow-up study 
Breast Cancer Research  2006;8(6):R67.
Background
Mutations in the ataxia-telangiectasia mutated (ATM) and checkpoint kinase 2 (CHEK2) genes and amplification of the v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2) gene have been suggested to have an important role in breast cancer aetiology. However, whether common variation in these genes has a role in the development of breast cancer or breast cancer survival in humans is still not clear.
Methods
We performed a comprehensive haplotype analysis of the ATM, CHEK2 and ERBB2 genes in a Swedish population-based study, which included 1,579 breast cancer cases and 1,516 controls. We followed the cases for 8.5 years, on average, and retrieved information on the date and cause of death during that period from the nationwide Swedish causes of death registry. We selected seven haplotype-tagging SNPs (tagSNPs) in the ATM gene, six tagSNPs in the CHEK2 gene and seven tagSNPs in the ERBB2 gene that predicted both haplotypic and single locus variations in the respective genes with R2 values ≥ 0.8. These tagSNPs were genotyped in the complete set of cases and controls. We computed expected haplotype dosages of the tagSNP haplotypes and included the dosages as explanatory variables in Cox proportional hazards or logistic regression models.
Results
We found no association between any genetic variation in the ATM, CHEK2 or ERBB2 genes and breast cancer survival or the risk of developing tumours with certain characteristics.
Conclusion
Our results indicate that common variants in the ATM, CHEK2 or ERBB2 genes are not involved in modifying breast cancer survival or the risk of tumour-characteristic-defined breast cancer.
doi:10.1186/bcr1623
PMCID: PMC1797028  PMID: 17132159

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