Generation of laterality depends on a pathway which involves the asymmetrically expressed genes nodal, Ebaf, Leftb, and Pitx2. In mouse, node monocilia are required upstream of the nodal cascade. In chick and frog, gap junctions are essential prior to node/organizer formation. It was hypothesized that differential activity of ion channels gives rise to unidirectional transfer through gap junctions, resulting in asymmetric gene expression. PKD2, which if mutated causes autosomal dominant polycystic kidney disease (ADPKD) in humans, encodes the calcium release channel polycystin-2. We have generated a knockout allele of Pkd2 in mouse. In addition to malformations described previously, homozygous mutant embryos showed right pulmonary isomerism, randomization of embryonic turning, heart looping, and abdominal situs. Leftb and nodal were not expressed in the left lateral plate mesoderm (LPM), and Ebaf was absent from floorplate. Pitx2 was bilaterally expressed in posterior LPM but absent anteriorly. Pkd2 was ubiquitously expressed at headfold and early somite stages, with higher levels in floorplate and notochord. The embryonic midline, however, was present, and normal levels of Foxa2 and shh were expressed, suggesting that polycystin-2 acts downstream or in parallel to shh and upstream of the nodal cascade.

Determination of the vertebrate left-right body axis during embryogenesis results in asymmetric development and placement of most inner organs. Although the asymmetric Nodal cascade is conserved in all vertebrates, the mechanism of symmetry breakage has remained controversial. In mammalian and fish embryos, a cilia-driven leftward flow of extracellular fluid is required for initiation of the Nodal cascade. This flow is localized at the posterior notochord ("node") and Kupffer's vesicle, respectively. In frog and chick embryos, however, molecular asymmetries are required earlier, from cleavage stages through gastrulation. The validity of a cilia-based mechanism for all vertebrates therefore has been questioned. Here we show that a cilia-driven leftward flow precedes asymmetric nodal expression in the frog Xenopus. Motile monocilia emerged on the gastrocoel roof plate during neurulation and lengthened and polarized from an initially central position to the posterior pole of cells. Concomitantly, a robust leftward fluid flow developed from stage 15 onward, significantly before asymmetric nodal transcription started in the left-lateral-plate mesoderm at stage 19. Injection of 1.5% methylcellulose into the archenteron prevented leftward flow and resulted in laterality defects, demonstrating that the flow itself was required for asymmetric gene expression and organ placement.

Background-Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves. Methods and Results-To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2 Ϫ/Ϫ embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos. Conclusions-From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.

The pharmacokinetics and safety of the L-valyl ester pro-drug of acyclovir, valaciclovir (256U87), were investigated in two phase I, placebo-controlled trials in normal volunteers. These included a single-dose study with doses from 100 to 1000 mg (single cohort) and a multiple-dose investigation with doses from 250 to 2000 mg (five separate cohorts). In each cohort, eight subjects received valaciclovir and four subjects received placebo. Pharmacokinetic findings for valaciclovir and acyclovir were consistent in the two studies. Valaciclovir was rapidly and extensively converted to acyclovir, resulting in significantly greater acyclovir bioavailability (approximately threefold to fivefold) compared with that historically observed with high-dose (800 mg) oral acyclovir. At the higher valaciclovir doses, acyclovir maximum concentration and daily area under the concentration-time curve approximated those obtained with intravenous acyclovir. The favorable safety profile and enhanced acyclovir bioavailability from valaciclovir administration has prompted additional clinical evaluations for zoster and herpes simplex virus treatment, as well as cytomegalovirus suppression in immunocompromised patients.

In vertebrates, the readily apparent left-right (L/R) anatomical asymmetries of the internal organs can be traced to molecular events initiated at or near the time of gastrulation. However, the earliest steps of this process do not seem to be universally conserved. In particular, how this axis is first defined in chicks has remained problematic. Here we show that asymmetric cell rearrangements take place within chick embryos, creating a leftward movement of cells around the node. It is the relative displacement of cells expressing Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) that is responsible for establishing their asymmetric expression patterns. The creation of asymmetric expression domains as a passive effect of cell movements represents an alternative strategy for breaking L/R symmetry in gene activity.In mice and rabbits monocilia responsible found on cells of the posterior notochordal plate have been shown to play a crucial role in breaking L/R symmetry (1,2). These cilia are able to create a leftward flow of fluid, in a pit-like teardrop shaped space that is not covered by subjacent endoderm (3)., The flow of fluid across this pit stimulates signal transduction that ultimately leads to induction of asymmetric gene expression (1,2).In the chick embryo, in contrast, the endoderm underlying Hensen's node (a structure at the rostral end of the primitive streak in the gastrulating embryo) exists as a continuous sheet ventral to the notochordal plate mesoderm (4) and there is no morphological pit on the ventral surface in which a flow of fluid could be established. Prior work has noted cilia at Hensen's node (5) but these short cilia are on endodermal cells and are unrelated to the motile cilia on the mesodermal cells of the ventral node in the mouse and rabbit. The mesodermal cells at Hensen's node in the chick are devoid of cilia. In addition, the Talpid chick mutant lacks primary cilia (6) but does not exhibit L/R asymmetry defects. Unlike the mouse and rabbit, the chick node itself becomes morphologically asymmetric, with a marked tilt towards the left around the time the primitive streak reaches full extension, at Stage 4. (7,8) (Fig. 1A-C). Shortly thereafter, a number of small L/R asymmetric expression domains are observed to the right and left of the node (9). However, all of the genes expressed in such a manner are initially expressed in a symmetric fashion, for example, Fgf8 bilaterally along the primitive streak and Shh bilaterally across the top of the node until stage 4, (10) (Fig. 1D_G) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript morphological asymmetries in the node, these gene expression patterns also become gradually asymmetric by stage 5 (Fig. 1H,I).To investigate cellular rearrangements that could be responsible for establishing the morphological asymmetry of the node, we performed a time lapse analysis of cell movements at Hensen's node; randomly labeling cells by electroporation of a green fluorescent protein (GFP) reporter. At stage 4, ...

N-Acetylation by hepatic arylamine N-acetyltransferase (NAT, EC 2.3.1.5) is a major route in the metabolism and detoxification of numerous drugs and foreign chemicals. NAT is the target of a common genetic polymorphism of clinical relevance in human populations. We have used our recently isolated rabbit cDNA rnat to clone three human NAT genes from human leukocyte DNA. None of the three genomic coding sequences was interrupted by introns. Two genes, designated NAT1 and NAT2, each possessed open reading frames of 870 bp. Both genes have been assigned to human chromosome 8, pter-q11. Following transfection they were transiently expressed in monkey kidney COS-1 cells. NAT1 and NAT2 gave rise to functional NAT proteins, as judged by their NAT enzyme activity with the arylamine substrate sulfamethazine. Western blots with NAT-specific antisera detected proteins of apparent molecular weight of 33 and 31 kD in NAT1- and NAT2-transfected cultures, respectively. The product of NAT2 had an identical apparent molecular weight as that of NAT detected in human liver cytosol. The deduced amino acid sequence of NAT2 also contained 6 peptide sequences which had previously been determined from tryptic peptides of the polymorphic NAT purified from human liver. These data suggest that NAT2 encodes the polymorphic NAT protein. The third gene, NATP, had multiple deleterious mutations and did not encode a functional NAT protein; it most likely represents a pseudogene.

No abstract

The acetylation polymorphism is one of the most common genetic variations in the transformation of drugs and chemicals. More than 50% of individuals in Caucasian populations are homozygous for a recessive trait and are of the "slow acetylator" phenotype. They are less efficient than "rapid acetylators" in the metabolism of numerous drugs and environmental and industrial chemicals. The acetylation polymorphism is associated with an increased risk of drug toxicity and with an increased frequency of certain cancers. We report the identification of the primary mutations in two alleles of the gene for the N-acetyltransferase (NAT; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) isozyme NAT2 associated with slow acetylation. These alleles, Ml and M2, account for more than 90% of slow acetylator alleles in the European population we have studied. Ml and M2 were identified by restriction fragment length polymorphisms with Kpn I and Msp I and subsequently cloned and sequenced. Ml and M2 each are characterized by a combination of two different point mutations, one causing an amino acid substitution (Ile-113 -* Thr in Ml, Gin in M2), the other being silent (C 481 -+ T in Ml, C 282 T in M2). Functional expression of Ml and M2 and of chimeric gene constructs between mutant and wild-type NAT2 in COS-1 cells suggests that Ml causes a decrease of NAT2 protein in the liver by defective translation, whereas M2 produces an unstable enzyme. On the basis of the mutations described here and a rare mutant allele (M3) reported recently, we have developed a simple DNA amplification assay that allows the predictive genotyping of more than 95% of slow and rapid acetylator alleles and the identification of individuals at risk.The acetylation polymorphism is one of the most common inherited variations in the biotransformation of drugs and chemicals. Its association with drug toxicity and an increased risk to develop certain cancers has made it one of the oldest and best-studied examples of a pharmacogenetic condition (1-3). Forty to 70% of Caucasians in Europe and North America are of the "slow acetylator" phenotype and are less efficient than "rapid acetylators" in the metabolism of numerous drugs and chemicals containing primary aromatic amine or hydrazine groups. These include agents such as isoniazid, sulfamethazine (SMZ) and other sulfonamides, procainamide, hydralazine, dapsone, and caffeine, as well as several chemicals with carcinogenic potential such as benzidine, 2-aminofluorene, and f-naphthylamine, present in dyes, antioxidants, pesticides, and explosives (2-4). Highly mutagenic and carcinogenic arylamines also are generated during cooking of food (5). Slow acetylators are at higher risk to develop bladder cancer (1-4), whereas rapid acetylators are at higher risk for colorectal cancer (6).In previous studies, we have shown that slow acetylators have a quantitative decrease in their liver of a cytosolic arylamine N-acetyltransferase (NAT; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) (7). This enzyme was p...