Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified.     

The stereochemistry of benzo[a]pyrene-2'-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)-trans-B[a]P-N(2)-dG and, to a lesser extent, (+)-cis-B[a]P-N(2)-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N(2)-dG and (+)-trans-B[a]P-N(2)-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA-protein complexes. We have previously shown that the (+)-trans-isomeric adduct inhibits both the binding and methylating efficiencies (k(cat)) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142-6159]. Here we show that the stereoisomeric (+)-cis-B[a]P-N(2)-dG lesion has only a minimal effect on the binding of these MTases and on k(cat). The minor-groove (+)-trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)-cis adduct does not interfere with the minor-groove DNA-catalytic loop contacts, allowing near-normal binding of the MTases and undiminished k(cat) values.     

Molecular genetics of attention-deficit hyperactivity disorder (ADHD): an update

Attention-deficit hyperactivity disorder (ADHD) is a heritable and behavioral condition of childhood, affecting 5-10% of school-age children worldwide. Affected patients exhibit various behavioral problems such as carelessness, restlessness, disobedience and failure to stay quiet in class. The etiology of ADHD is not known. However, family, twin and adoption studies have provided strong evidence for a genetic etiology of the disorder. A genome-wide scan has identified six chromosomal loci with LOD scores suggestive of linkage. Animal studies suggest the involvement of the brain dopamine pathway and its alteration in ADHD but there is no direct evidence to support this hypothesis. In addition, there are at least 20 candidate genes of small effect that have been studied but none of them appear to be the major gene causing ADHD. Medical intervention along with psychosocial therapy proved to be beneficial for controlling ADHD, although some undesirable side effects have been encountered during medical treatment. In the future, identification of environmental factors, study of additive gene effects and the interaction of genes and environmental factors may provide better insight into the pathophysiology of ADHD. This may lead to an effective new treatment strategy.     

Hereditary degenerative retinopathies: optimism for somatic gene therapy

Retinitis pigmentosa comprises a large and exceptionally heterogeneous group of hereditary disorders of the retina. As a result of an extensive investigation around the world, primary genetic lesions have been described in many genes. Some of these genes encode enzymes that are involved in the signal transduction pathway. On the basis of in vitro functional assays and standard transgenic and knock-out experiments, it has been proposed that normal cell functions are disrupted because of an abnormal protein-folding and metabolic errors or structural defects in the membrane. This ultimately leads to a gene-mediated cell death known as apoptosis. Various gene transfer approaches using mouse models further suggest that the degeneration can be rescued to some extent. Although many questions remain to be answered, investigations during the last 10 years have enormously increased our understanding of this exceptionally heterogeneous disorder and give hope for an effective gene therapy and a possible cure.     

A 5-base insertion in the γC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract

A seven-generation family with 30 members affected by highly variable autosomal dominant zonular pulverulent cataracts has been previously described. We have localized the cataracts to a 19-cM interval on chromosome 2q33-q35 including the gamma-crystallin gene cluster. Maximum lod scores are 4.56 (theta=0.02) with D2S157, 3.66 (theta=0.12) with D2S72, and 3.57 (theta=0.052) with CRYG. Sequencing and allele-specific oligonucleotide analysis of the pseudo gammaE-crystallin promoter region from individuals in the pedigree suggest that activation of the gammaE-crystallin pseudo gene is unlikely to cause the cataracts in the family. In addition, base changes in the TATA box but not the Sp1-binding site have been found in unaffected controls and can be excluded as a sole cause of cataracts. In order to investigate the underlying genetic mechanism of cataracts in this family further, exons of the highly expressed gammaC- and gammaD-crystallin genes have been sequenced. The gammaD-crystallin gene shows no abnormalities, but a 5-bp duplication within exon 2 of the gammaC-crystallin gene has been found in one allele of each affected family member and is absent from both unaffected family members and unaffected controls. This mutation disrupts the reading frame of the gammaC-crystallin coding sequence and is predicted to result in the synthesis of an unstable gammaC-crystallin with 38 amino acids of the first "Greek key" motif followed by 52 random amino acids. This finding suggests that the appropriate association of mutant betagamma-crystallins into oligomers is not necessary to cause cataracts and may give us new insights into the genetic mechanism of cataract formation.     

Evaluation of the prothrombin gene polymorphism in patients with advanced retinopathy of prematurity

It has been reported recently that a common genetic variant in the 3'-untranslated region of the prothrombin gene is associated with a significant fraction of premature births. The purpose of this study is to evaluate the prothrombin gene polymorphism in a large cohort of patients with preterm birth and advanced retinopathy of prematurity. For this purpose, the leukocyte DNAs were analyzed for the mutation (20210A) in the 3'-untranslated region of the prothrombin gene by PCR amplification, followed by restriction analysis and DNA sequencing. Our extensive analysis revealed a normal genotype (GG) in all patients as well as controls. These results suggest that the common genetic variant in the 3'-untranslated region of the prothrombin gene is not associated with advanced retinopathy of prematurity. Although more patients' samples should be evaluated, this genetic test does not support a relationship between prothrombin gene mutation and retinopathy of prematurity.     

Mammalian cochlear genes and hereditary deafness

Deafness is the most common sensory hereditary disorder. It is a genetically heterogeneous and multifactorial disease affecting approximately 1 infant in 2000. It can be acquired or congenital and can also be syndromic or nonsyndromic. There are approximately 70 genetic loci that have been described for nonsyndromic deafness in humans and 25 auditory-pigmentary diseases in mice. The past 2 years have witnessed remarkable progress in identifying the genes involved in both syndromic and nonsyndromic disorders in humans and mice. Many of these are expressed in the inner ear and are most likely involved in cochlear physiology and development. However, the phenotypic variability in patients carrying the same genetic change, and discrepancies between the phenotypes of mice and humans carrying the same gene defect, emphasize environmental factors and interacting genes in producing the clinical outcome. In the future, molecular understanding of the etiology of the disorder may lead to a cure or delay the onset of the disorder.     

A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale.

A continuous-flow capillary mixing apparatus, based on the original design of Regenfuss et al. (Regenfuss, P., R. M. Clegg, M. J. Fulwyler, F. J. Barrantes, and T. M. Jovin. 1985. Rev. Sci. Instrum. 56:283-290), has been developed with significant advances in mixer design, detection method and data analysis. To overcome the problems associated with the free-flowing jet used for observation in the original design (instability, optical artifacts due to scattering, poor definition of the geometry), the solution emerging from the capillary is injected directly into a flow-cell joined to the tip of the outer capillary via a ground-glass joint. The reaction kinetics are followed by measuring fluorescence versus distance downstream from the mixer, using an Hg(Xe) arc lamp for excitation and a digital camera with a UV-sensitized CCD detector for detection. Test reactions involving fluorescent dyes indicate that mixing is completed within 15 micros of its initiation and that the dead time of the measurement is 45 +/- 5 micros, which represents a >30-fold improvement in time resolution over conventional stopped-flow instruments. The high sensitivity and linearity of the CCD camera have been instrumental in obtaining artifact-free kinetic data over the time window from approximately 45 micros to a few milliseconds with signal-to-noise levels comparable to those of conventional methods. The scope of the method is discussed and illustrated with an example of a protein folding reaction.