Background. Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene.Methods. To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls.Results. Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent.Conclusions. Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD. The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men. (N Engl

Preclinical studies have shown that gene transfer following readministration of viral vectors is often inefficient due to the presence of neutralizing antibodies. Vectors derived from ubiquitous human adenoviruses may have limited clinical use because preexisting humoral and cellular immunity is found in 90% of the population. Furthermore, risks associated with the use of human adenovirus vectors, such as the need to immunosuppress or tolerize patients to a potentially debilitating virus, are avoidable if efficient nonhuman adenovirus vectors are feasible. Plasmids containing recombinant canine adenovirus (CAV) vectors from which the E1 region had been deleted were generated and transfected into a CAV E1-transcomplementing cell line. Vector stocks, with titers greater than or equal to those obtained with human adenovirus vectors, were free of detectable levels of replication-competent CAV and had a low particle-to-transduction unit ratio. CAV vectors were replication defective in all cell lines tested, transduced human-derived cells at an efficiency similar to that of a comparable human adenovirus type 5 vector, and are amenable to in vivo use. Importantly, 49 of 50 serum samples from healthy individuals did not contain detectable levels of neutralizing CAV antibodies.Human adenovirus types 2 and 5 were chosen as potential gene transfer vectors because of the significant amount of research performed on these serotypes. However, vectors derived from viruses that naturally infect and replicate in humans may not be the optimal candidates for therapeutic applications. Adenoviruses are ubiquitous in all populations and can be lethal in infants and immunocompromised patients (5, 18, 24). More than 90% of the adult population has detectable levels of circulating antibodies directed against antigens from human serotypes (9, 32, 33). Phase I trials using human adenovirus vectors have yielded conflicting results (8,21,41). A difference in humoral immunity that is directed against the vector capsid might explain, in addition to other factors, the variability between and within these studies. Furthermore, when repeat administrations were attempted (7, 42), transgene activity was not detected. Studies aimed at immunotolerization of mice, for the primary or repeat delivery of human adenovirus vectors, are interesting from the immunological standpoint but may have limited practical use in the clinic. Will immunotolerization of patients to adenovirus vectors activate latent, more virulent serotypes? Concomitantly, there are other drawbacks associated with human-derived adenovirus vectors. More than 95% of a healthy cohort had a long-lived CD4 ϩ T-cell response directed against multiple human adenovirus serotypes (14). These data imply that adenovirus serotype switching (27) may have limited advantages. Furthermore, replication-competent adenoviruses (RCAs) (26) can potentially contaminate human adenovirus-derived vector stocks, including gutless adenovirus vectors (16,22), while E1 region-positive vectors are a potential contam...

We have investigated the lignin peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. Pre-steady-state kinetic analyses demonstrated that guaiacol is a good substrate for both compounds I and II, the two-and one-electron oxidized enzyme intermediates, respectively, of lignin peroxidase. The rate constant for the reaction with compound I is 1.2 ؋ 10 6 M ؊1 s ؊1. The reaction of guaiacol with compound II exhibits a K d of 64 M and a first-order rate constant of 17 s ؊1 . Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with a K m of 160 M and a k cat of 7.7 s ؊1. Veratryl alcohol, a secondary metabolite of ligninolytic fungi, is capable of mediating the oxidation of guaiacol. This was shown by steady-state inhibition studies. Guaiacol completely inhibited the oxidation of veratryl alcohol, whereas veratryl alcohol had no corresponding inhibitory effect on guaiacol oxidation. In fact, at low guaiacol concentrations, veratryl alcohol stimulated the rate of guaiacol oxidation. These results collectively demonstrate that veratryl alcohol can serve as a mediator for phenolic substrates in the lignin peroxidase reaction.This study investigates the ability of 3,4-dimethoxybenzyl (veratryl) alcohol to mediate the lignin peroxidase-catalyzed oxidation of guaiacol. Lignin peroxidases are hemeproteins secreted by the white rot fungus Phanerochaete chrysosporium during secondary metabolism (1, 2). These isozymes catalyze the oxidation of lignin and a large number of phenolic and non-phenolic substrates (3, 4). The catalytic cycle of lignin peroxidase is similar to that of other peroxidases (5, 6) where ferric enzyme is first oxidized by H 2 O 2 to generate the twoelectron oxidized intermediate, compound I (7). Compound I is then reduced by one electron donated by a substrate molecule, yielding the 1-electron oxidized enzyme intermediate, compound II, and a free radical product. The catalytic cycle is completed by the one-electron reduction of compound II by a second substrate molecule.In the absence of a reducing substrate, the enzyme can undergo a series of reactions with H 2 O 2 to form compound III, oxyperoxidase (6,8). It is also well documented that prolonged incubation of enzyme with H 2 O 2 in the absence of a reducing substrate such as veratryl alcohol can cause irreversible inactivation of the enzyme (9). In the presence of veratryl alcohol, however, lignin peroxidase undergoes multiple turnovers without any detectable inactivation. Because veratryl alcohol is normally produced by ligninolytic cultures of P. chrysosporium (10), workers have proposed that its physiological function is to protect the enzyme from H 2 O 2 -dependent inactivation (11).An alternate role for veratryl alcohol in lignin biodegradation has been proposed by Harvey et al. (12). These workers observed that substrates that are not oxidized by lignin peroxidase such as anisyl alcohol and 4-methoxyman...