Cloning of cDNAs encoding xenopus neuregulin: expression in myotomal muscle during embryo development

The ontogeny of the myotome was investigated using [3H]thymidine or Brdu treatment in conjunction with 1,1', di-octadecyl-3, 3, 3', 3',-tetramethylindo-carbocyanine perchlorate (DiI) labeling and expression of specific markers. We have identified a subset of early post-mitotic cells that is present in the dorsomedial aspect of epithelial somites and is homogeneously distributed along their entire rostrocaudal extent. The post-mitotic quality of this cell subset enabled us to trace their fate in time-course experiments. Following initial somite dissociation, this epithelial post-mitotic layer bends underneath the medial portion of the nascent dermomyotome. Then, these cells progressively lose epithelial arrangement and migrate in a rostral direction where they accumulate temporarily. Subsequently, these early post-mitotic precursors extend processes that reach both rostral and caudal edges of each segment. Medial somite-derived myofibers also fill the entire mediolateral extent of the segment and reach the dorsomedial lip of the dermomyotome, thus forming the primary myotome. During this process, their large nuclei localize to a narrow stripe in the middle of the nascent myotome. Consistent with the proliferation studies, DiI labeling of the medial epithelial somite cells gave rise to a primary myotomal structure, and continuous pulsing of the DiI-injected embryos with radioactive thymidine revealed that these fibers indeed developed from post-mitotic progenitors. As these early post-mitotic cells that arise prior to somite dissociation are the first wave of progenitors that constitutes the myotome, we have termed them avian muscle pioneers. We propose that the primary myotome formed by the muscle pioneers constitutes a longitudinal scaffold that serves as a substrate for the addition of subsequent waves of myotomal cells.     

The relationship between arm-ankle pressure difference and peak systolic velocity in patients with stenotic lower extremity vein grafts

The relationship between the measured arm-ankle pressure difference (AAPD), or the ankle/arm index (AAI), and the focal peak systolic velocity (PSV) at stenotic sites of infrainguinal vein grafts has not been determined. We attempted to relate these two parameters. We used Doppler systolic pressures and duplex ultrasonography to study 35 infrainguinal vein bypass grafts followed in a surveillance protocol. The following graft groups were identified: grafts in nondiabetic patients (n = 26), grafts in diabetic patients (n = 9), nonrevised stenotic grafts (n = 14), revised stenotic grafts (n = 14), and normal grafts (n = 7). AAPD and AAI were measured in both lower extremities. Pressure gradients across graft stenoses were indirectly estimated using the modified Bernoulli equation (delta P =4V2). Measured AAPDs and estimated pressure gradients showed moderate correlation in nondiabetic (r = 0.58) and diabetic (r = 0.63) patients. Correlation was fair (r = 0.3) prior to graft revision. There was no correlation (r = 0.1) in the nonrevised stenotic grafts. For individual patients with stenotic grafts who were followed in consecutive visits, the correlation varied from none to good (r range 0.01 to 0.71). We conclude that there is a lack of consistent correlation between the measured AAPD, or AAI, and the estimated stenotic graft pressure gradient. This finding illustrates the limitation of the AAI as a monitoring test to predict failure of stenotic infrainguinal vein grafts.     

Effect of length of gestation on maternal cellular immunity to human trophoblast antigens

Maternal lymphocyte reactivity to human trophoblast antigens was studied in placentas of gestational ages 8 to 14 weeks and 32 to 34 weeks, respectively. Significant trophoblast lysis became apparent after 24 hours' incubation in the latter case compared with a time lag of 72 hours in the terminated gestations. Maternal cellular immunity, therefore, was not detected during the first 3 1/2 months of pregnancy, but was detectable by the time of parturition. The possible significance is discussed with respect to the antigenic stimulus and survival of the fetal allograft.     

The yeast telomere length counting machinery is sensitive to sequences at the telomere-nontelomere junction

Saccharomyces cerevisiae telomeres consist of a continuous 325 +/- 75-bp tract of the heterogeneous repeat TG1-3 which contains irregularly spaced, high-affinity sites for the protein Rap1p. Yeast cells monitor or count the number of telomeric Rap1p molecules in a negative feedback mechanism which modulates telomere length. To investigate the mechanism by which Rap1p molecules are counted, the continuous telomeric TG1-3 sequences were divided into internal TG1-3 sequences and a terminal tract separated by nontelomeric spacers of different lengths. While all of the internal sequences were counted as part of the terminal tract across a 38-bp spacer, a 138-bp disruption completely prevented the internal TG1-3 sequences from being considered part of the telomere and defined the terminal tract as a discrete entity separate from the subtelomeric sequences. We also used regularly spaced arrays of six Rap1p sites internal to the terminal TG1-3 repeats to show that each Rap1p molecule was counted as about 19 bp of TG1-3 in vivo and that cells could count Rap1p molecules with different spacings between tandem sites. As previous in vitro experiments had shown that telomeric Rap1p sites occur about once every 18 bp, all Rap1p molecules at the junction of telomeric and nontelomeric chromatin (the telomere-nontelomere junction) must participate in telomere length measurement. The conserved arrangement of these six Rap1p molecules at the telomere-nontelomere junction in independent transformants also caused the elongated TG1-3 tracts to be maintained at nearly identical lengths, showing that sequences at the telomere-nontelomere junction had an effect on length regulation. These results can be explained by a model in which telomeres beyond a threshold length form a folded structure that links the chromosome terminus to the telomere-nontelomere junction and prevents telomere elongation.     

trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP

Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.     

Activation of particulate guanylyl cyclase by Vibrio vulnificus hemolysin

Recently we reported that Vibrio vulnificus hemolysin, an exotoxin produced by V. vulnificus, dilates rat thoracic aorta via elevated cGMP levels without affecting nitric oxide synthase. We investigated the mechanism further by observing the guanylyl cyclase activities in cytosolic, membrane, unfractionated, or reconstituted preparations. Hemolysin did not activate guanylyl cyclase in the membrane or cytosolic fraction, while it activated guanylyl cyclase in unfractionated or reconstituted preparation. The increased activity was not inhibited by the HS-142-1, a microbial polysaccharide which antagonizes atrial natriuretic peptide receptor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor. However, it was attenuated by 6-(phenylamino)-5,8-quinolinedione (LY 83.583), which inhibits the catalytic domain of both guanylyl cyclases, and by cholesterol, which blocks hemolysin-incorporation into the membrane. Removing ATP, a cofactor of particulate guanylyl cyclase, attenuated the activation and ATPgammaS, a non-phosphorylating analog, restored it. These results suggest that V. vulnificus hemolysin activates particulate guanylyl cyclase via hemolysin incorporation into the vascular smooth muscle cell membrane in cooperation with certain unidentified cytosolic component(s).     

Isolation of synthetic lethal mutants of ras1 in Schizosaccharomyces pombe

Ras proteins are membrane-associated guanine nucleotide-binding proteins that serve as molecular switches for signal transduction pathways in a diverse array of organisms. Various cellular factors are known to interact with Ras proteins. In order to find the novel cellular factors that are associated with Ras function, we have constructed synthetic lethal mutants of the ras1+ gene in Schizosaccharomyces pombe and used them to identify the genes that are functionally dependent on the Ras1. We first constructed S. pombe strains in which chromosomal ras1+ gene is placed under the nmt1 promoter that is regulated by thiamine. This strain shows ras1+ phenotype in the absence of thiamine, whereas it shows ras1- phenotype in the presence of thiamine. Second, we mutated the constructed strains with ultraviolet light (UV) and selected two synthetic lethal mutants that could not grow when Ras1 function was repressed (ras1-). One of the mutants, KSC3, showed a swollen cell shape, aberrant deposition of septum materials, and aberrant nuclei. The other mutant, KSC4, showed sensitivity to hyper-osmolarity when Ras1 function is absent. These mutants, however, grow normally when Ras1 is expressed (ras1+). These two novel synthetic lethal mutants of ras1 provide the means to isolate the corresponding genes that function in association with Ras1 in S. pombe. Screening of a genomic library of S. pombe complementing the mutant phenotype allowed us to identify several novel genes associated with Ras1 of S. pombe.