Mediastinitis due to Gram-negative bacteria is associated with increased mortality.

The aim of this study was to describe the features of a large cohort of patients with postoperative mediastinitis, with particular regard to Gram-negative bacteria (GNB), and assess their outcome. This bicentric retrospective cohort included all patients who were hospitalized in the Intensive Care Unit with mediastinitis after cardiac surgery during a 9-year period. Three hundred and nine patients developed a mediastinitis with a mean age of 65 years and a mean standard Euroscore of six points. Ninety-one patients (29.4%) developed a GNB mediastinitis (GNBm). Of the 364 pathogens involved, 103 GNB were identified. GNBm were more frequently polymicrobial (44% versus 3.2%; p <0.001). Being female was the sole independent risk factor of GNBm in multivariate analysis. Initial antimicrobial therapy was significantly more frequently inappropriate with GNBm compared with other microorganisms (24.6% versus 1.9%; p <0.001). Independent risk factors for inappropriateness of initial antimicrobial treatment were GNBm (OR = 8.58, 95%CI 2.53-29.02, p 0.0006), and polymicrobial mediastinitis (OR = 4.52, 95%CI 1.68-12.12, p 0.0028). GNBm were associated with more drainage failure, secondary infection, need for prolonged mechanical ventilation and/or use of vasopressors. Thirty-day hospital mortality was significantly higher with GNBm (31.9 % versus 17.0%; p 0.004). GNBm was identified as an independent risk factor of hospital mortality (OR = 2.31, 95%CI 1.16-4.61, p 0.0179).

Net1 stimulates RNA polymerase I transcription and regulates nucleolar structure independently of controlling mitotic exit.

The budding yeast RENT complex, consisting of at least three proteins (Net1, Cdc14, Sir2), is anchored to the nucleolus by Net1. RENT controls mitotic exit, nucleolar silencing, and nucleolar localization of Nop1. Here, we report two new functions of Net1. First, Net1 directly binds Pol I and stimulates rRNA synthesis both in vitro and in vivo. Second, Net1 modulates nucleolar structure by regulating rDNA morphology and proper localization of multiple nucleolar antigens, including Pol I. Importantly, we show that the nucleolar and previously described cell cycle functions of the RENT complex can be uncoupled by a dominant mutant allele of CDC14. The independent functions of Net1 link a key event in the cell cycle to nucleolar processes that are fundamental to cell growth.

Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast.

In the budding yeast Saccharomyces cerevisiae, the multifunctional protein Net1 is implicated in regulating the cell cycle function of the Cdc14 protein phosphatase. Genetic and cell biological data suggest that during interphase and early mitosis Net1 holds Cdc14 within the nucleolus where its activity is suppressed. Upon its transient release from Net1 at late anaphase, active Cdc14 promotes exit from mitosis by dephosphorylating targets in the nucleus and cytoplasm. In this paper we present evidence supporting the proposed role of Net1 in regulating Cdc14 and exit from mitosis. We show that the NH(2)-terminal fragment Net1(1-600) directly binds Cdc14 in vitro and is a highly specific competitive inhibitor of its activity (K(i) = 3 nm) with five different substrates including the physiologic targets Swi5 and Sic1. An analysis of truncation mutants indicates that the Cdc14 binding site is located within a segment of Net1 containing residues 1-341. We propose that Net1 inhibits by occluding the active site of Cdc14 because it acts as a competitive inhibitor, binds to a site located within the catalytic domain (residues 1-374), binds with reduced affinity to a Cdc14 C283S mutant in which an active site Cys is replaced, and is displaced by tungstate, a transition state analog known to bind in the catalytic site of protein-tyrosine phosphatases.

Human sex ratio at birth and mother's birth season: multivariate analysis.

We used a population-based historical French Canadian database to examine the effects of mother's birth season on sex ratio at birth. Non-first births in the database (n = 127,658) were analyzed for their sex, parish size (2 large parishes of Montreal and Quebec or the other smaller parishes), time period (births up to 1719 or those from 1720), maternal age (< or = 24, 25-29, 30-34, 35+ years), sex of the preceding sibling (male or female), and birth seasons of the child and his or her parents (February-April, May-July, August-October, November-January). Season of child's birth significantly affected the sex ratio (chi 2 = 11.507, d.f. = 3, p = 0.009), with the births in February-April or May-July showing a lower sex ratio. Season of mother's birth also contributed highly significantly to the variation of sex ratio (chi 2 = 15.196, d.f. = 3, p = 0.002); mothers born in February-April had a low sex ratio among their children (sex ratio = 1.013). In contrast, season of father's birth did not affect the sex ratio (chi 2 = 0.618, d.f. = 3, p = 0.892). When a multiple logistic model was applied to the data, mother's birth season was the single most significant factor. The lower sex ratio from mothers born in February-April was observed consistently for every maternal age and delivery season. Seasonal influences on female fetuses seem to have changed their future reproductive characteristics.

The tetratricopeptide repeat domain and a C-terminal region control the activity of Ser/Thr protein phosphatase 5.

Protein Ser/Thr phosphatase 5 is a 58-kDa protein containing a catalytic domain structurally related to the catalytic subunits of protein phosphatases 1, 2A, and 2B and an extended N-terminal domain with three tetratricopeptide repeats. The activity of this enzyme is stimulated 4-14-fold in vitro by polyunsaturated fatty acids and anionic phospholipids. The structural basis for lipid activation of protein phosphatase 5 was examined by limited proteolysis and site-directed mutagenesis. Trypsinolysis removed the tetratricopeptide repeat domain and increased activity to approximately half that of lipid-stimulated, full-length enzyme. Subtilisin removed the tetratricopeptide repeat domain and 10 residues from the C terminus, creating a catalytic fragment with activity that was equal to or greater than that of lipid-stimulated, full-length enzyme. Catalytic fragments generated by proteolysis were no longer stimulated by lipid, and degradation of the tetratricopeptide repeat domain was decreased by association with lipid. A truncated mutant missing 13 C-terminal residues was also insensitive to lipid and was as active as full-length, lipid-stimulated enzyme. These results suggest that the C-terminal and N-terminal domain act in a coordinated manner to suppress the activity of protein phosphatase 5 and mediate its activation by lipid. These regions may be targets for the regulation of protein phosphatase 5 activity in vivo.

Human PIR1 of the protein-tyrosine phosphatase superfamily has RNA 5'-triphosphatase and diphosphatase activities.

A human cDNA was isolated encoding a protein with significant sequence similarity (41% identity) to the BVP RNA 5'-phosphatase from the Autographa californica nuclear polyhedrosis virus. This protein is a member of the protein-tyrosine phosphatase (PTP) superfamily and is identical to PIR1, shown by Yuan et al. (Yuan, Y., Da-Ming, L., and Sun, H. (1998) J. Biol. Chem. 272, 20347-20353) to be a nuclear protein that can associate with RNA or ribonucleoprotein complexes. We demonstrate that PIR1 removes two phosphates from the 5'-triphosphate end of RNA, but not from mononucleotide triphosphates. The specific activity of PIR1 with RNA is several orders of magnitude greater than that with the best protein substrates examined, suggesting that RNA is its physiological substrate. A 120-amino acid segment C-terminal to the PTP domain is not required for RNA phosphatase activity. We propose that PIR1 and its closest homologs, which include the metazoan mRNA capping enzymes, constitute a subgroup of the PTP family that use RNA as a substrate.

Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex.

Exit from mitosis in budding yeast requires a group of essential proteins--including the GTPase Tem1 and the protein phosphatase Cdc14--that downregulate cyclin-dependent kinase activity. We identified a mutation, net1-1, that bypasses the lethality of tem1 delta. NET1 encodes a novel protein, and mass spectrometric analysis reveals that it is a key component of a multifunctional complex, denoted RENT (for regulator of nucleolar silencing and telophase), that also contains Cdc14 and the silencing regulator Sir2. From G1 through anaphase, RENT localizes to the nucleolus, and Cdc14 activity is inhibited by Net1. In late anaphase, Cdc14 dissociates from RENT, disperses throughout the cell in a Tem1-dependent manner, and ultimately triggers mitotic exit. Nucleolar sequestration may be a general mechanism for the regulation of diverse biological processes.

A protein tyrosine phosphatase-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activities.

The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5'-phosphatase. BVP sequentially removes gamma and beta phosphates from the 5' end of triphosphate-terminated RNA, leaving a 5'-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA.

Marriage season, promptness of successful pregnancy and first-born sex ratio in a historical natural fertility population--evidence for sex-dependent early pregnancy loss?

We investigated population-based vital records of the seventeenth and eighteenth century French Canadian population to assess the effects of marriage season on the outcome of the first births under natural fertility conditions (n = 21,698 marriages). Promptness of the first successful conception after marriage differed according to marriage season; the proportion of marriages with a marriage-first birth interval of 8.0-10.0 months was lowest (34%) for marriages in August-October (P = 0.001). Although the male/female sex ratio of the babies born with an interval of 8.0-10.0 months was generally higher (1.10) than those with an interval of 10.0-24.0 months (1.05), the marriages in August-October resulted in a significantly reduced sex ratio (0.96) among only the prompt conceptions (P = 0.026). We discuss whether this seasonal reduction of the sex ratio could be partly explained by a clustered pregnancy loss of male zygotes in early pregnancy.

High refractive errors and the accident/incident rate in Canadian medical category 1 pilots.

BACKGROUND: Since 1982, the Canadian Civil Aviation Medicine Division has medically certified to Category 1 standard commercial and airline transport pilots whose visual correction was in excess of +/- 3.5 diopters (D). METHOD: A review between the years 1982 and 1991 of the 253 pilots who had been medically certified, although they were outside the standard, was conducted. We determined if there was any difference in the accident/incident rate in this group as compared with the Canadian general aviation population standardized to a rate per 100,000 flying hours. The 253 pilots were divided into two groups with Group A having a refractive error outside the range +/- 5.7 D and Group B having a refractive error range of +/- 3.5 to +/- 5.6 D. RESULTS: The Group A rate was within the expected range of accidents and incidents per 100,000 flying hours. The accident/incident rate in Group B was significantly lower than the expected average. CONCLUSION: In conclusion, the Canadian Civil Aviation Medicine Division's policy on granting "flexibility" to applicants with moderate to high refractive errors has not affected adversely the accident or incident rate and therefore has not compromised aviation safety.

The noncatalytic C-terminal segment of the T cell protein tyrosine phosphatase regulates activity via an intramolecular mechanism.

Human T cell protein tyrosine phosphatase (TCPTP) is a nontransmembrane enzyme, the first of the protein tyrosine phosphatase family to be cloned. Alternative mRNA splicing results in variation in the sequence at the extreme C terminus of TCPTP and generates a 45-kDa form (TC45) that is targeted to the nucleus and a 48-kDa variant (TC48) associated with membranes of the endoplasmic reticulum. In this report, we assessed the role of the C-terminal, noncatalytic segment of TCPTP in regulating activity, concentrating primarily on the TC45 variant. We have demonstrated that limited tryptic proteolysis of TC45 releases first a 42-kDa fragment, then a 33-kDa catalytic domain. Using reduced carboxyamidomethylated and maleylated lysozyme as substrate (RCML), the catalytic domain displays 20-100-fold more activity than the full-length enzyme. Analysis of the time course of limited trypsinolysis revealed that proteolytic activation occurred following cleavage of a protease-sensitive region (residues 353-387) located at the C terminus of TC45. The activity of truncation mutants illustrated that removal of 20 C-terminal residues was sufficient to activate the enzyme fully. The 33-kDa catalytic domain, but not the full-length enzyme, was inhibited in a concentration-dependent manner by addition of the noncatalytic C-terminal segment of TC45. A monoclonal antibody to TCPTP, CF4, which recognizes an epitope located between residues 350 and 363, was capable of fully activating TC45. These data indicate that the noncatalytic segment of TC45 contains an autoregulatory site that modulates activity via a reversible intramolecular interaction with the catalytic domain. These studies suggest that the C-terminal noncatalytic segment of TC45, and possibly TC48, may not only direct the enzyme to different subcellular locations but may also modulate activity in response to the binding of regulatory proteins and/or posttranslational modification.

The activity of Cdc14p, an oligomeric dual specificity protein phosphatase from Saccharomyces cerevisiae, is required for cell cycle progression.

The essential CDC14 gene of the budding yeast, Saccharomyces cerevisiae, encodes a 62-kDa protein containing a sequence that conforms to the active site motif found in all enzymes of the protein tyrosine phosphatase superfamily. Genetic studies suggest that Cdc14p may be involved in the initiation of DNA replication, but its precise cell cycle function is unknown. Recombinant Cdc14p was produced in bacteria, characterized, and shown to be a dual specificity protein phosphatase. Polyanions such as polyglutamate and double-stranded and single-stranded DNA bind to Cdc14p and affect its activity. Native molecular weights of 131,000 and 169,000 determined by two independent methods indicate that recombinant Cdc14p self-associates in vitro to form active oligomers. The catalytically inactive Cdc14p C283S/R289A mutant is not able to suppress the temperature sensitivity of a cdc14-1(ts) mutant nor replace the wild type gene in vivo, demonstrating that phosphatase activity is required for the cell cycle function of Cdc14p. A distinctive COOH-terminal segment (residues 375-551) is rich in Asn and Ser residues, carries a net positive charge, and contains two tandem 21-residue repeats. This COOH-terminal segment is not required for activity, for oligomerization, or for the critical cell cycle function of Cdc14p.

Purification of a fatty acid-stimulated protein-serine/threonine phosphatase from bovine brain and its identification as a homolog of protein phosphatase 5.

An arachidonic acid-stimulated Ser/Thr phosphatase activity was detected in soluble extracts prepared from rat pituitary clonal GH4C1 cells, rat or bovine brain, and bovine heart. The enzyme activity was purified to homogeneity from bovine brain as a monomer with a Mr of 63,000 and a specific activity of 32 nmol of Pi released per min/mg of protein when assayed in the presence of 10 microM phosphocasein in the absence of lipid. Arachidonic acid stimulated activity 4-14-fold, with half-maximal stimulation at 50-100 microM, when assayed in the presence of a variety of phosphosubstrates including casein, reduced carboxamidomethylated and maleylated lysozyme, myelin basic protein, and histone. Oleic acid, linoleic acid, and palmitoleic acid also stimulated activity; however, saturated fatty acids and alcohol or methyl ester derivatives of fatty acids did not significantly affect activity. The lipid-stimulated phosphatase was identified as the bovine equivalent of protein phosphatase 5 or a closely related homolog by sequence analysis of proteolytic fragments generated from the purified enzyme. When recombinant rat protein phosphatase 5 was expressed as a cleavable glutathione S-transferase fusion protein, the affinity-purified thrombin-cleaved enzyme exhibited a specific activity and sensitivity to arachidonic acid similar to those of the purified bovine brain enzyme. These results suggest that protein phosphatase 5 may be regulated in vivo by a lipid second messenger or another endogenous activator.

Season of birth and Alzheimer's disease: a population-based study in Saguenay-Lac-St-Jean/Québec (IMAGE Project).

The birth distribution of 399 cases of Alzheimer's disease (AD) identified in the region of Saguenay-Lac-St-Jean (Québec) was compared with that of: (a) the population currently living in the area; and (b) the population born during the same period in the same area. AD cases have been recruited since 1986 by the IMAGE Project. Cases and controls were grouped according to the month of birth and according to the day of birth using density estimation. Analyses showed a significant deficit of births in the month of May. We believe these preliminary results deserve further attention and we suggest two possible explanations that could lead to a deficit of AD births at specific periods during the year.

Identification of inhibitory and calmodulin-binding domains of the PDE1A1 and PDE1A2 calmodulin-stimulated cyclic nucleotide phosphodiesterases.

Using a bovine 61-kDa (PDE1A2) calmodulin-stimulated phosphodiesterase (CaM-PDE) cDNA and a bovine lung 59-kDa (PDE1A1) CaM-PDE cDNA reported here, we have identified two new regions within the primary structure of these two related isozymes that are important for regulation by Ca2+/CaM. PDE1A1 is identical to the PDE1A2 isozyme except for the amino-terminal 18 residues. In agreement with earlier studies, the CaM concentration required for half-maximal activation (KCaM) of recombinant PDE1A1 (0.3 nM) was approximately 10-fold less than the KCaM for recombinant PDE1A2 (4 nM). A series of deletion mutations of the PDE1A2 cDNA removing nucleotide sequence encoding the first 46-106 aminoterminal residues were constructed and expressed using the baculovirus system. Deletion of the amino acids encompassing a previously identified, putative CaM-binding domain (residues 4-46) produced a polypeptide that was still activated 3-fold by CaM (KCaM approximately 3 nM). However, complete CaM-independent activation occurred when residues 4-98 were deleted. To determine the location of the additional CaM-binding domain(s), the inhibitory potency of seven overlapping, synthetic peptides spanning amino acids 76-149 of PDE1A2 was tested using the CaM-activated enzyme. One peptide spanning amino acids 114-137 of PDE1A2 appeared to be the most potent inhibitor of CaM-stimulated activity. These results reveal the existence of a CaM-binding domain located approximately 90 residues carboxyl-terminal to the putative CaM-binding domains previously identified within the PDE1A1 and PDE1A2 isozymes. Moreover, a discrete segment important for holding these CaM-PDEs in a less active state at low Ca2+ concentrations is located between the two CaM-binding domains.

Slow twin conception at first birth and subsequent maternal twin proneness in a natural fertility population.

To study whether apparently more fecund women having delivered twins at first birth have traits of higher twin-proneness, we performed a retrospective cohort study on population-based historical vital records of the 17-18th century French Canadian immigrants and their descendants under natural fertility conditions. Among 24896 mothers who had at least one child, 248 had twin maternities at their first birth (twinning rate = 1.0%). Among 21508 mothers with a valid marriage-first birth interval, twinning rate was 0.97% among prompt conceptions (7.0-11.0 months), with a particularly high rate at the interval of 7.0-8.0 months (2.2%). Marriages in August-October resulted in a higher twinning rate particularly for the slow conceptions than those in the other seasons. Promptly-conceived mothers of twins at the first delivery may seem to have higher fecundity, but subsequent births from these mothers (n = 88) show a lower twinning rate (1.7%) particularly at younger maternal age than from the other mothers who had slowly conceived twins at their first birth (n = 112). The latter show a 4.5% twinning rate as a whole among their second or later births. So-called twin-proneness of a mother, whether genetic or acquired, was not connected to higher conception rate of twin's mothers immediately after marriage. Reduced fecundity, which may have been imposed by some environmental factors, could raise the chance of twinning.

Reproductive life of French-Canadians in the 17-18th centuries: a search for a trade-off between early fecundity and longevity.

One of the predictions derived from Williams' (1957) evolutionary theory of senescence is the existence of a trade-off between early fecundity and longevity. The population register of the French immigrants to Québec in the 17th century and of the first Canadians in the 17th and 18th centuries was used to detect such a trade-off in a noncontraceptive human population living at a time when longevity had not been prolonged by medical care and was not artificially shortened by wars, epidemics, or other external causes. No evidence for such a trade-off could be detected in these populations which had not yet reached the demographic transition phase (i.e., the historical period when longevity began to be extended and the progeny began to be reduced). Results are discussed in connection with the various studies aiming to test the Williams' theory.

Identification of sites for feedback regulation of glutamine 5-phosphoribosylpyrophosphate amidotransferase by nucleotides and relationship to residues important for catalysis.

Glutamine phosphoribosylpyrophosphate amidotransferase, the key regulatory enzyme for de novo purine nucleotide synthesis, is subject to feedback regulation by adenine and guanine nucleotides. Affinity labeling with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-monophosphate (N3-AMP) was used to identify purine nucleotide sites for feedback control of the Escherichia coli amidotransferase. FSBA inactivated the amidotransferase with saturation kinetics. Specificity for inactivation was shown by the covalent attachment of 2.0-2.4 eq of [3H] sulfobenzoyladenosine (SBA) per subunit and protection by GMP and AMP against inactivation and incorporation of [3H]SBA. Six chymotryptic peptides modified with [3H]SBA were isolated and identified by differential labeling followed by high performance liquid chromatography and radioactivity. Mass spectrometry and Edman degradation analysis were used to identify 5 residues that were covalently modified by [3H]SBA: Tyr74, Tyr258, Lys326, Tyr329, and Tyr465. Tyr258 was also modified by N3-AMP. Mutant enzymes K326Q and Y329A had activity similar to that of the wild type enzyme. However, both mutants exhibited decreased sensitivity to inhibition by GMP and decreased binding of GMP but were inhibited by AMP. Mutant enzymes Y74A and Y258F were normally feedback-inhibited but were defective in glutamine amide transfer and synthase functions, respectively. Therefore Tyr74 and Tyr258 are important for activity and modification by FSBA and N3-AMP accounts for enzyme inactivation. These results localize residues important for catalysis in close proximity to a site for nucleotide binding. Two additional mutant enzymes, G331I and N351A, were constructed which were refractory to inhibition by GMP with little change in inhibition by AMP. A replacement of Tyr465 indicates that this residue is not essential for catalysis or feedback inhibition. Overall, these results are interpreted in terms of a two-nucleotide site model with Lys326, Tyr329, Gly331, and Asn351 defining a site required for inhibition by GMP. A second nucleotide site not affinity labeled by analogs is very close to or overlaps with the catalytic site.

The baculovirus Autographa californica encodes a protein tyrosine phosphatase.

The genome of the baculovirus Autographa californica encodes a 19-kDa protein (BVP) containing an active site sequence motif ((I/V)HCXAGXXR(S/T)G) that characterizes a large family of protein tyrosine phosphatases (PTPs). The baculoviral protein was expressed in Escherichia coli and purified so that its enzymatic properties could be examined. We have demonstrated that recombinant BVP has intrinsic protein tyrosine phosphatase activity. Like VH1, a PTP encoded by vaccinia virus, BVP also dephosphorylates seryl or threonyl residues. However, the similarity of BVP to VH1 or the catalytic domains from PTPs of eukaryotic origin is restricted to a small region surrounding the active site motif. In contrast, the similarity of BVP to two putative PTPs encoded by the CDC14 gene of Saccharomyces cerevisiae and a gene of unknown function from Caenorhabditis elegans extends throughout its sequence. We postulate that BVP and its two homologs constitute a unique subfamily that may differ from other PTPs in having a specialized function, mode of regulation, or substrate preference.