The oral nitrate-reducing capacity correlates with peak power output and peak oxygen uptake in healthy humans.

Interest in inorganic nitrate and nitrite has grown substantially over the past decade as research has revealed the role of these anions in enhancing nitric oxide (NO) availability through an oral pathway. Nitrite synthesis in the mouth seems to be an important mechanism to feed the circulatory system with this anion. This is interesting since greater plasma nitrite concentration has been associated with better fitness levels in humans, but this question has not been investigated in relation to salivary nitrite concentration. Additionally, no previous study has investigated the oral nitrate-reducing capacity in regards to peak oxygen uptake (VO2peak) or peak power output (Wpeak) in humans. Thus, the main goal of this study was to investigate whether salivary nitrite and nitrate concentration and the oral nitrate-reducing capacity were associated with VO2peak and Wpeak in healthy humans. Fifty individuals (22 females and 28 males; 38.8 ±â€¯14.3 years/old; BMI = 22.8 ±â€¯3.9) performed a graded exercise test on a cycle ergometer to assess their VO2peak and Wpeak. Unstimulated salivary samples were taken before and 20 min after exercise to measure nitrate/nitrite, pH and lactate. The oral nitrate-reducing capacity was also assessed in 25 subjects before and after exercise. Oral nitrate-reducing capacity was positively associated with Wpeak (rs = 0.64; P = 0.001) and the VO2peak (rs = 0.54; P = 0.005). Similar correlations were found when these variables were analysed after exercise. In addition, a significant decrease in salivary pH (pre: 7.28 ±â€¯0.361; post-exercise: 7.16 ±â€¯0.33; P = 0.003) accompanied by an increase of salivary lactate (pre: 0.17 ±â€¯0.14 mmol/L; post-exercise: 0.48 ±â€¯0.38; P < 0.001) was found after exercise. However, these changes did not have any impact on salivary nitrate/nitrite concentration and the oral nitrate-reducing capacity after exercise. In conclusion, this is the first evidence showing a link between the oral nitrate-reducing capacity and markers of aerobic fitness levels in healthy humans.

Comparison of identical temperature-sensitive mutations in the L polymerase proteins of sendai and parainfluenza3 viruses.

The L subunit of the RNA-dependent RNA polymerase of negative strand RNA viruses is believed to possess all the enzymatic activities necessary for viral transcription and replication. Mutations in the L proteins of human parainfluenza virus type 3 (PIV3) and vesicular stomatitis virus (VSV) have been shown to confer temperature sensitivity to the viruses; however, their specific defects have not been determined. Mutant PIV3 L proteins expressed from plasmids were tested for temperature sensitivity in transcription and replication in a minigenome reporter system in cells and for in vitro transcription from purified PIV3 template. The single L mutants, Y942H and L992F, were temperature sensitive (ts) in both assays, although viral RNA synthesis was not completely abolished at the nonpermissive temperature. Surprisingly, the T1558I L mutant was not ts, although its cognate virus was ts. Thus the ts defect in this virus may be due to the abrogation of an essential interaction of the mutant polymerase with a host cell component, which is not measured by the RNA synthesis assays. Most of the combinations of the PIV3 L mutations were not additive and did not show temperature sensitivity in in vitro transcription. Since they were ts in the minigenome assay in vivo, replication appears to be specifically defective. The ts mutations in PIV3 and VSV L proteins were also substituted into the Sendai L protein to compare the defects in related systems. Only Sendai Y942H L was ts in both transcription and replication. One Sendai L mutant, L992F, gave much better replication than transcription. Several other mutants could transcribe but not replicate in vitro, while replication in vivo was normal.

Mutations in conserved domains IV and VI of the large (L) subunit of the sendai virus RNA polymerase give a spectrum of defective RNA synthesis phenotypes.

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein. When aligned with amino acid sequences of L proteins from other negative-sense RNA viruses, the Sendai L protein contains six regions of good conservation, designated domains I-VI, which have been postulated to be important for the various enzymatic activities of the polymerase. To directly address the roles of domains IV and VI, 14 site-directed mutations were constructed either by changing clustered charged amino acids to ala or by substituting selected Sendai L amino acids with the corresponding sequence from measles virus L. Each mutant L protein was tested for its ability to transcribe and replicate the Sendai genome. The series of mutations created a spectrum of phenotypes, from those with significant, near wild-type, activity to those being completely defective for all RNA synthesis. The inactive L proteins, however, were still able to bind P protein and form a polymerase capable of binding the nucleocapsid template. The remainder of the mutations reduced, but did not abolish, enzymatic activity and included one mutant with a specific defect in the synthesis of the leader RNA compared with mRNA, and three mutants that replicated genome RNA much more efficiently in vivo than in vitro. Together, these data suggest that even within a domain, the function of the Sendai L protein is likely to be very complex. In addition, SS3 and SS10 L in domain IV and SS13 L in domain VI were shown to be temperature-sensitive. Both SS3 and SS10 gave significant, although not wild-type, activity at 32 degrees C; however, each was completely inactivated for all RNA synthesis at 37 and 39.6 degrees C. SS13 was completely inactive only when synthesized at the higher temperature. Each polymerase synthesized at 32 degrees C could only be partially heat inactivated in vitro at 39.6 degrees C, suggesting that inactivation involves both thermal lability of the protein and temperature sensitivity for its synthesis.

Mutations in conserved domain II of the large (L) subunit of the Sendai virus RNA polymerase abolish RNA synthesis.

The large (L) protein of Sendai virus complexes with the phosphoprotein (P) to form the active RNA-dependent RNA polymerase. The L protein is believed to be responsible for all of the catalytic activities of the polymerase associated with transcription and replication. Sequence alignment of the L proteins of negative-strand RNA viruses has revealed six conserved domains (I-VI) thought to be responsible for the enzymatic activities. Charged-to-alanine mutagenesis was carried out in a highly charged, conserved region (amino acids 533-569) within domain II to test the hypothesis of Müller et al. [J. Gen. Virol. 75, 1345-1352 (1994)] that this region may contribute to the template binding domain of the viral RNA polymerase. The mutant proteins were tested for expression and stability, the ability to synthesize viral RNA in vitro and in vivo, and protein-protein interactions. Five of the seven mutants were completely defective in all viral RNA synthesis, whereas two mutants showed significant levels of both mRNA and leader RNA synthesis. One of the transcriptionally active mutants also gave genome replication in vitro although not in vivo. The other mutant was defective in all the replication assays and thus the mutation uncoupled transcription and replication. Because the completely inactive L mutants can bind to the P protein to form the polymerase complex and the polymerases bind to the viral nucleocapsid template, these amino acids are essential for the activity of the L protein.

Dissection of individual functions of the Sendai virus phosphoprotein in transcription.

The Sendai virus P protein is an essential component of the viral RNA polymerase (P-L complex) required for RNA synthesis. To identify amino acids important for P-L binding, site-directed mutagenesis of the P gene changed 17 charged amino acids, singly or in groups, and two serines to alanine within the L binding domain from amino acids 408 to 479. Each of the 10 mutants was wild type for P-L and P-P protein interactions and for binding of the P-L complex to the nucleocapsid template, yet six showed a significant inhibition of in vitro mRNA and leader RNA synthesis. To determine if binding was instead hydrophobic in nature, five conserved hydrophobic amino acids in this region were also mutated. Each of these P mutants also retained the ability to bind to L, to itself, and to the template, but two gave a severe decrease in mRNA and leader RNA synthesis. Since all of the mutants still bound L, the data suggest that L binding occurs on a surface of P with a complex tertiary structure. Wild-type biological activity could be restored for defective polymerase complexes containing two P mutants by the addition of wild-type P protein alone, while the activity of two others could not be rescued. Gradient sedimentation analyses showed that rescue was not due to exchange of the wild-type and mutant P proteins within the P-L complex. Mutants which gave a defective RNA synthesis phenotype and could not be rescued by P establish an as-yet-unknown role for P within the polymerase complex, while the mutants which could be rescued define regions required for a P protein function independent of polymerase function.

Identification of nucleocapsid protein residues required for Sendai virus nucleocapsid formation and genome replication.

Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP0-P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.

The Sendai virus C protein binds the L polymerase protein to inhibit viral RNA synthesis.

The Sendai virus nested set of C proteins which are expressed in an alternative open reading frame from the P mRNA has been shown to downregulate viral RNA synthesis. Utilizing a glutathione S-transferase (gst) C fusion protein (gstC), we have shown that C protein forms a complex with the L, but not the P, subunit of the viral RNA polymerase. When P, L, and gstC are coexpressed, an oligomer of P, through its interaction with L, is also bound to beads. Since binding of C to L in the P-L complex does not disrupt P binding, the C and P binding sites appear to be different. GstC binding to L occurs only when the proteins are coexpressed in the same cell. The gstC, but not gst, protein inhibits viral transcription in vitro, showing that the fusion protein retains biological function. Pulse-chase experiments of the various complexes show that L protein synthesized alone has a half-life of 1. 2 hr, which is increased 12.5-fold by binding P, but is not significantly increased by binding gstC. Analyses of complex formation with truncations of L protein show that the C-terminal 1333 amino acids of L are not required for binding C. The dose-response curves show that replication of the genomic DI-H RNA is more sensitive to inhibition by C protein than is the synthesis of DI leader RNA, suggesting that the downregulation of RNA synthesis may be more complex than just the inhibition of the initiation of RNA synthesis.

The Sendai virus V protein interacts with the NP protein to regulate viral genome RNA replication.

The interactions of Sendai virus proteins required for viral RNA synthesis have been characterized both by the yeast two-hybrid system and through the use of glutathione S-transferase (gst)-viral fusion proteins synthesized in mammalian cells. Using the two-hybrid system we have confirmed the previously identified P-L (RNA polymerase), NPo-P (encapsidation substrate), and P-P complexes and now demonstrate NP-NP and NPo-V protein interactions. Expression of gstP and P proteins and binding to glutathione-Sepharose beads as a measure of complex formation confirmed the P-P interaction. The P-gstP binding occurred only on expression of the proteins in the same cell and was mapped to amino acids 345-411. We also show that full-length and deletion gstV and gstW proteins bound NPo protein when these sets of proteins were coexpressed and have identified one required region from amino acids 78-316. Neither gstV nor gstW bound NP assembled into nucleocapsids. Furthermore, both V and W proteins lacking the N-terminal 77 amino acids inhibited DI-H genome replication in vitro, showing the biological relevance of the remaining region. We propose that the specific inhibition of genome replication by V and W proteins occurs through interference with either the formation or the use of the NPo-P encapsidation substrate.

Mutations in conserved domain I of the Sendai virus L polymerase protein uncouple transcription and replication.

To begin to map functional domains of the Sendai P-L RNA polymerase complex we wanted to characterize the P binding site on the Sendai L protein. Analysis of in vitro and in vivo P-L polymerase complex formation with carboxyl-truncations of the L protein showed that the N-terminal half of the protein was required. Site-directed mutagenesis of the Sendai virus L gene was employed to change amino acids within a highly conserved region of the N-terminal domain I from amino acids (aa) 348-379 singly or in pairs from the Sendai to the corresponding measles L sequence or to alanine. The mutant L proteins coexpressed with the viral P and NP proteins in mammalian cells were assayed for their ability to form the P-L complex and to synthesize RNA in vitro and showed a variety of defective phenotypes. While most of the mutant L proteins still formed the P-L polymerase complex, a change from serine to arginine at aa 368 and a three-amino-acid insertion at aa 379 virtually abolished both complex formation and RNA synthesis. Changes of aas 370 and 376-377 in the L protein gave only small decreases in viral RNA synthesis. Substitutions at either aas 349-350 or aas 354-355 and a three-amino-acid insertion at aa 348 in the L protein yielded enzymes that catalyzed significant transcription, but were defective in DI RNA replication, thus differentially affecting the two processes. Since DI leader RNA, but not genome RNA, was still synthesized by this class of mutants, the defect in replication appears to be in the ability of the mutant enzyme to package newly synthesized nascent RNA. Single changes at aas 362, 363, and 366 in the L protein gave enzymes with severely decreased overall RNA synthesis, although some leader RNA was synthesized, suggesting that they cannot transcribe or replicate past the leader gene. These studies have identified a region in conserved domain I critical for multiple functions of the Sendai virus L protein.

An amino-proximal domain of the L protein binds to the P protein in the measles virus RNA polymerase complex.

The RNA polymerase of measles virus consists of two virus-encoded subunits, the L and P proteins with 2183 and 507 amino acids, respectively. When these proteins were coexpressed from plasmids in a mammalian expression system, a complex was formed as detected by the coimmunoprecipitation of the L protein with the P protein by anti-P antibodies. Pulse-chase experiments showed that complex formation increased the stability of the L protein. We have used the coimmunoprecipitation assay in conjunction with a series of C-terminal truncations of the L protein to map the region of the L protein which is involved in complex formation with the P protein. Mutant L proteins consisting of the N-terminal 1139, 916, 511, and 408 amino acids all bound to the P protein. An L protein truncation consisting of only the N-terminal 292 amino acids, which deleted part of the conserved domain I, however, did not bind the P protein. The data show that the N-terminal 408 amino acids of the L protein contain the P binding domain and suggest that domain I within this region of the L proteins of (-) strand RNA viruses may be important for RNA polymerase complex formation.

Deletion analysis defines a carboxyl-proximal region of Sendai virus P protein that binds to the polymerase L protein.

The Sendai virus RNA polymerase complex consists of two viral proteins, L and P, which must be coexpressed in order to form the active enzyme. Pulse-chase experiments show that the L protein is unstable when synthesized in the absence of the P protein, but is stable in the P-L complex. Using sequential deletions in the P protein (568 amino acids), we have mapped the site on the P protein where the L protein binds by co-immunoprecipitation and gradient sedimentation analyses. The L-binding site residues in the C-terminal half of the P protein, since deletion of up to amino acid 324 of P protein does not affect complex formation. The L-binding site was mapped to a region of P protein encompassing amino acids 412-478. This region lies between the previously mapped amino acid regions which form the nucleocapsid-binding domain (amino acids 345-411 and 479-568). The data suggest that the L and NP protein-binding domains on P protein do not overlap.

Determinants of serotype specificity in transcription of vesicular stomatitis virus synthetic nucleocapsids.

Transcription of the nucleocapsid template of vesicular stomatitis virus (VSV) is serotype specific, since the viral RNA polymerase of the VSV Indiana (Ind) serotype transcribes the Ind nucleocapsid but not the nucleocapsid of the VSV New Jersey (NJ) serotype and, similarly, the NJ RNA polymerase transcribes only the NJ nucleocapsid. We have prepared synthetic nucleocapsid templates from various combinations of purified leader gene RNA and N protein of these two VSV serotypes for analysis of serotype specificity in vitro. In agreement with previous observations, in vitro transcription of synthetic homologous nucleocapsids with the 3' terminal leader RNA gene of either the (+) or (-) strand sense and the N protein of the same serotype is also serotype specific. We show with chimeric nucleocapsids, where the RNA and N protein are of different serotypes, that both components of the template are important for specificity, although the specifics depend on the serotype from which the RNA polymerase is derived. The Ind RNA polymerase will transcribe only the homologous nucleocapsids where both the RNA and N protein are of the Ind serotype, and not the chimeric nucleocapsids. In contrast, the NJ RNA polymerase is active not only on the homologous synthetic nucleocapsid, but also gives significant levels of transcription as long as one of the nucleocapsid components (N protein or RNA) is of the NJ serotype. The divergent RNA sequence of the distal portion of the leader gene (nt22 to 50/51) seems to be a major determinant for specificity, since transcription of synthetic nucleocapsids containing just the conserved proximal 1-22 nucleotides is significantly less serotype-restricted. Restriction appears to occur at the level of elongation of the product rather than initiation of RNA synthesis, since synthesis of template-length RNA, but not reiterated small initiation products, is inhibited. In addition, the serotype of the P protein subunit of the RNA polymerase also contributes to the serotype specificity of transcription, since the NJ P protein can bind equally to NJ and Ind nucleocapsids, while Ind P protein binds preferentially to Ind nucleocapsids.

Promoter analysis of the vesicular stomatitis virus RNA polymerase.

We have reconstituted synthetic VSV nucleocapsids from synthetic RNA and purified VSV N protein which are active as templates for transcription by the viral RNA polymerase in vitro. Utilizing progressively shorter RNAs in this system, we found that nucleocapsids containing just the 3' 22 nucleotides of either (-) or (+) strand viral RNA served as good transcription templates. Of this sequence, optimal transcription required the 15-17 3'-terminal nucleotides as a promoter. We showed with two VSV serotypes that the 3'-terminal nucleotides, UGC, were absolutely essential for transcription of the synthetic nucleocapsid by added RNA polymerase. Addition of extra nucleotides to the normal 3' end of nucleocapsid RNA totally abolished activity. The requirements for the nucleotides from positions 4 to 17 from the 3' end were less stringent, since individual changes in this region had variable effects on transcription, from partial inhibition to stimulation. The characterization of the transcription products synthesized from a variety of nucleocapids showed both full-length products and short (8-12 nucleotides) products which initiated directly at the 3' end but prematurely terminated. The VSV RNA polymerase was capable of adding one or more nontemplated bases to the 3' end of the product RNA depending on the nucleocapsid RNA sequence and the presence or absence of 5' triphosphates on the RNA.

Preparing children for surgery. Learning through play.

The approach that Children's Hospital has taken in preparing children for surgery, both ambulatory and extended recovery, is exciting and challenging. The staff is encouraged by the positive response of the patients and families who participate in the tour and the doll demonstrations.

Serum and peritoneal fluid amylase levels in CAPD. Normal values and clinical usefulness.

The mean serum amylase of 42 asymptomatic CAPD patients was elevated but was not significantly different from that of a group of chronic hemodialysis patients. Serum amylase levels in CAPD patients with peritonitis were not elevated with respect to asymptomatic patients. Amylase activity was not detectable in the peritoneal fluid of 38/42 asymptomatic patients and 6/13 peritonitis patients and was present at low levels in the other 11 patients. Patients with other abdominal conditions (pancreatitis, cholecystitis and small bowel perforation) had very marked elevations of serum and/or peritoneal fluid amylase which differentiated them from the asymptomatic and peritonitis patients. Although hyperamylasemia is common in asymptomatic CAPD patients and in those with peritonitis, measurement of serum and peritoneal fluid amylase levels is useful in the evaluation of CAPD patients presenting with abdominal symptoms.

Characterization of VP-16-induced DNA damage in isolated nuclei from L1210 cells.

Based on the observation that VP-16-induced DNA damage can be demonstrated in isolated nuclei but not in purified DNA, and that this effect is temperature-dependent, it is postulated that the mechanism of action of VP-16 involves an essential intranuclear event, perhaps enzyme-mediated, which is a prerequisite for the cleavage of DNA. Using alkaline elution to assay single-strand breaks in isolated L1210 nuclei, we have further characterized conditions influencing this putative intranuclear reaction. We have found drug activity to be dependent on magnesium and pH and to be stimulated by low concentrations of ATP (0.05-1 mM), an effect which was not observed with a nonhydrolyzable analog of ATP. Heat-labile activity in a nuclear non-histone protein extract was critical to VP-16-mediated DNA damage. This new evidence lends further credence to the hypothesis that activity of an intranuclear enzyme, possessing characteristics consistent with a type II DNA topoisomerase, is a prerequisite for the cleavage of DNA by VP-16.