Influence of Phytase Transgenic Corn on the Intestinal Microflora and the Fate of Transgenic DNA and Protein in Digesta and Tissues of Broilers

An experiment was conducted to investigate the effect of phytase transgenic corn (PTC) on intestinal microflora, and the fate of transgenic DNA and protein in the digesta and tissues of broilers. A total of 160 1-day-old Arbor Acres commercial male broilers were randomly assigned to 20 cages (8 chicks per cage) with 10 cages (replicates) for each treatment. Birds were fed with a diet containing either PTC (54.0% during 1–21 days and 61.0% during 22–42 days) or non-transgenic isogenic control corn (CC) for a duration of 42 days. There were no significant differences (P>0.05) between birds fed with the PTC diets and those fed with the CC diets in the quantities of aerobic bacteria, anaerobic bacteria, colibacillus and lactobacilli, or microbial diversities in the contents of ileum and cecum. Transgenic phyA2 DNA was not detected, but phyA2 protein was detected in the digesta of duodenum and jejunum of broilers fed with the PTC diets. Both transgenic phyA2 DNA and protein fragments were not found in the digesta of the ileum and rectum, heart, liver, kidney, and breast or thigh muscles of broilers fed with the PTC diets. It was concluded that PTC had no adverse effect on the quantity and diversity of gut microorganisms; Transgenic phyA2 DNA or protein was rapidly degraded in the intestinal tract and was not transferred to the tissues of broilers.     

A novel common missense mutation G301C in the N-acetylgalactosamine-6-sulfate sulfatase gene in mucopolysaccharidosis IVA

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by a genetic defect in N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In previous studies, we have found two common mutations in Caucasians and Japanese, respectively. To characterize the mutational spectrum in various ethnic groups, mutations in the GALNS gene in Colombian MPS IVA patients were investigated, and genetic backgrounds were extensively analyzed to identify racial origin, based on mitochondrial DNA (mtDNA) lineages. Three novel missense mutations never identified previously in other populations and found in 16 out of 19 Colombian MPS IVA unrelated alleles account for 84.2% of the alleles in this study. The G301C and S162F mutations account for 68.4% and 10.5% of mutations, respectively, whereas the remaining F69V is limited to a single allele. The skewed prevalence of G301C in only Colombian patients and haplotype analysis by restriction fragment length polymorphisms in the GALNS gene suggest that G301C originated from a common ancestor. Investigation of the genetic background by means of mtDNA lineages indicate that all our patients are probably of native American descent. Received: 2 January 1997 / Accepted: 10 June 1997     

Left atrial appendage closure: outcomes and challenges

Whereas the left atrial appendage plays a rather minor role under physiological circumstances, it gains an importance in patients with atrial fibrillation. Compelling evidence has revealed that the left atrial appendage is implicated as the source of thrombus in the vast majority of strokes in atrial fibrillation. Oral anticoagulation remains the standard of care for stroke prevention in atrial fibrillation; nevertheless, this treatment has several limitations and is often contraindicated, particularly in the elderly population in whom the risk of stroke is high. Therefore, occluding the left atrial appendage is a logical approach to prevent thrombus formation and subsequent cardioembolic events in these patients. We present a review of clinical outcomes of patients with atrial fibrillation undergoing left atrial appendage closure and the challenges faced in this field.     

Visualization of HIV-1 Interactions with Penile and Foreskin Epithelia: Clues for Female-to-Male HIV Transmission

To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/− 0.0154 and 0.0171 +/− 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/− 0.0079 virions/image) than glans tissue (0.0167 +/− 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/− 0.0188 vs. 0.0151 +/− 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/− 3.908 vs. 12.466 +/− 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men.     

Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module

Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme.     

Low-level inversion of the L component of pseudorabies virus is not dependent on sequence homology.

Pseudorabies virus has a class 2 genome in which the S component is found in two orientations relative to the L component. The L component is bracketed by sequences that are partially homologous; it is found mainly in one orientation, but a small proportion is inverted (J. M. DeMarchi, Z. Lu, G. Rall, S. Kuperschmidt, and T. Ben-Porat, J. Virol. 64:4968-4977, 1990). We have ascertained the role of the patchy homologous sequences bracketing the L component in its inversion. A viral mutant, vYa, from which the sequences at the right end of the L component were deleted was constructed. Despite the absence of homologous sequences bracketing the L component in vYa, its L component inverted to an extent similar to that of the L component in the wild-type virus. These results show the following. (i) The low-frequency inversion of the L component of PrV is not mediated by homologous sequences bracketing this component. (ii) Cleavage of concatemeric DNA at the internal junction between the S and L components is responsible for the appearance of the minority of genomes with an inverted L component in populations of pseudorabies virus. (iii) The signals present near or at the end of the S component are sufficient to allow low-frequency cleavage of concatemeric DNA; the sequences at the end of the L component are not essential for cleavage, although they enhance it considerably.     

A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: application to the EcoRI and RsrI restriction and modification enzymes.

A modified oligodeoxyribonucleotide duplex containing an unnatural internucleotide trisubstituted 3' to 5' pyrophosphate bond in one strand [5'(oligo1)3'-P(OCH3)P-5'(oligo2) 3'] reacts with nucleophiles in aqueous media by acting as a phosphorylating affinity reagent. When interacted with a protein, a portion of the oligonucleotide [--P-5'(oligo2)3'] becomes attached to an amino acid nucleophilic group through a phosphate of the O-methyl-modified pyrophosphate linkage. We demonstrate the affinity labeling of nucleophilic groups at the active sites of the EcoRI and RsrI restriction and modification enzymes with an oligodeoxyribonucleotide duplex containing a modified scissile bond in the EcoRI recognition site. With the EcoRI and RsrI endonucleases in molar excess approximately 1% of the oligonucleotide becomes attached to the protein, and with the companion methyltransferases the yield approaches 40% for the EcoRI enzyme and 30% for the RsrI methyltransferase. Crosslinking proceeds only upon formation of a sequence-specific enzyme-DNA complex, and generates a covalent bond between the 3'-phosphate of the modified pyrophosphate in the substrate and a nucleophilic group at the active site of the enzyme. The reaction results in the elimination of an oligodeoxyribonucleotide remnant that contains the 3'-O-methylphosphate [5'(oligo1)3'-P(OCH3)] derived from the modified phosphate of the pyrophosphate linkage. Hydrolysis properties of the covalent protein-DNA adducts indicate that phosphoamide (P-N) bonds are formed with the EcoRI endonuclease and methyltransferase.