Mechanisms for secretion of extracellular proteins by gram-negative bacteria.
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Bacterial Proteins↗
Biomedical subjects
R A Welch
Publications and source records attributed to R A Welch.
Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion.
The primary structure of the Escherichia coli hemolysin polypeptide (HlyA) is used to predict intramolecular structures involved in the secretion and cytolytic activity of the molecule. The C-terminal region of HlyA contains a repeated, 8-amino acid chain represented by the consensus sequence Leu-Xaa-Gly-Gly-Xaa-Gly-Asn-Asp. Three in vitro derived mutations of hlyA are described that encode molecules missing various portions of the C-terminal region, including the repeat region. The wild-type and mutated HlyA molecules were analyzed for the ability to be secreted and to lyse erythrocytes. Hemolytic activity absolutely requires the presence of the repeats. The ability of the mutated HlyA molecules to initiate membrane translocation and be secreted required the presence of the C terminus and, to a degree, the repeated amino acid octets.
Amino Acid Sequence↗
Medical retirements and deaths in service of diabetic employees in the Post Office.
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Adolescent↗
Amniotic fluid thymosin alpha 1 levels increase during gestation.
Thymosin alpha 1 is one of several cytokines produced by the thymus that modulates immune function. The presence of elevated serum levels of thymosin alpha 1 in pregnant women and their newborns has suggested that this peptide may play a role in perinatal immunology. In this investigation, we used a microenzyme-linked immunosorbent assay to assay amniotic fluid for immunoreactive thymosin alpha 1 and found levels that were remarkably higher than newborn serum levels (P less than 10(-4]. The increase of thymosin alpha 1 in amniotic fluid with fetal age was natural logarithmic (r = 0.838, P less than 10(-6]. Thymosin alpha 1 in amniotic fluid may account for some of the immunologic properties of this medium.
Amniotic Fluid↗
Transcriptional organization of the Escherichia coli hemolysin genes.
The transcriptional organization of the Escherichia coli hemolysin genes (hlyCABD) encoded by pSF4000 was examined. The use of different hemolysin gene-specific radiolabeled probes in blots containing isolated in vivo RNA revealed 4.0-kilobase hlyCA and 8.0-kilobase hlyCABD transcripts. The treatment of cells with rifampin just before RNA isolation showed the half-lives of these mRNAs to be 10.2 and 4.4 min, respectively. The 5' ends of the hly transcripts were 462 and 464 nucleotides from the putative initiation codon of hlyC based on a primer extension method of RNA mapping. Deletion analysis of pSF4000 combined with quantification of the hemolysin structural protein HlyA by immunoblotting confirmed that major control of HlyA expression occurs within a 168-base-pair PstI fragment located 433 base pairs upstream of the start of hlyC. A second recombinant plasmid, pANN202-312, encoding an E. coli hemolysin of different origin expressed 6-fold less total HlyA and 50-fold less extracellular HlyA than pSF4000 in identical cell backgrounds. The pANN202-312 recombinant had a different hly promoter, with the hly mRNA beginning 264 nucleotides upstream from the start of hlyC. We showed by RNA blotting that cells harboring pANN202-312 compared with pSF4000 have similar steady-state levels of the hlyCA transcript but they lack a consistently detectable hlyCABD transcript. We propose that one reason for the disparate levels of extracellular hemolysin produced by hemolytic E. coli is dissimilar levels of mRNA encoding in part the transport genes hlyB and hlyD.
Base Sequence↗
Preeclampsia, delivery, and the hemostatic system.
To determine the effects of preeclampsia and delivery, the hemostatic system was evaluated before and 24 to 48 hours after delivery in 59 nulliparous patients without clinical signs of disseminated intravascular coagulation. Fifteen patients with mild preeclampsia and 18 with severe preeclampsia were compared with 26 pregnant control patients. Preeclampsia was associated with high fibronectin (p less than 0.001), low antithrombin III (p less than 0.001), and low alpha 2-antiplasmin (p less than 0.005), suggesting endothelial injury, clotting, and fibrinolysis, respectively. After delivery, fibronectin decreased only in preeclamptic patients (p less than 0.005); alpha 2-antiplasmin increased in all groups (p less than 0.001). Endothelial injury in preeclampsia appeared to resolve soon after delivery, which could contribute to the rapid clinical improvement noted in the early puerperium.
Adult↗
Employment of diabetics in a Post Office region.
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Diabetes Mellitus↗
Maternal and fetal circulating levels of thymosin alpha 1 during parturition.
The thymus directs T-lymphocyte development and contributes to the maintenance of immune homeostasis, in part, through its production of peptides known as thymosins. In pregnancy, maternal serum levels of thymosin alpha 1 have been reported to be low at midgestation and to increase by term, suggesting that maternal levels represent fetal levels. To evaluate this further, we obtained maternal venous and newborn mixed cord blood from 90 pregnancies between 20 and 42 weeks of gestation at delivery. An ELISA was used for thymosin alpha 1 assay, and analysis was by paired t test and regression. Maternal and newborn levels were independent of gestational age, but an apparent association (r = 0.51) between the two was inconclusive. Maternal levels (1,207 +/- 947 pg/ml) tended to be higher than those of healthy adults (1,043 +/- 576 pg/ml). Mixed umbilical cord serum levels (1,466 +/- 940 pg/ml) were higher than maternal levels (P less than or equal to 0.005). Although maternal thymosin alpha 1 levels may reflect fetal levels, immunological perturbations related to parturition appear to influence both.
Adolescent↗
Identification of two different hemolysin determinants in uropathogenic Proteus isolates.
DNA sequences similar to those of the Escherichia coli hemolysin genes were detected among uropathogenic isolates of Proteus vulgaris and Morganella morganii by using the Southern blotting technique and hly gene-specific DNA probe. Immunoblotting revealed that among the hemolytic P. vulgaris and M. morganii isolates there was expressed a polypeptide species similar in molecular size (110 kilodaltons) and antigenicity to Escherichia coli HlyA. A plasmid-mediated P. vulgaris hemolysin determinant identified by Southern blotting analysis was molecularly cloned, and the recombinant plasmid (pWPV100) was characterized by restriction endonuclease fragment mapping. A second recombinant library of genomic DNA prepared from a hemolytic, urinary tract isolate of Proteus mirabilis was constructed in E. coli. A 5.5-kilobase XhoI fragment encoding an extracellular hemolytic activity was molecularly cloned (pWPM100), and this plasmid was subjected to transposon-mediated mutagenesis with TnphoA. The P. mirabilis hemolytic phenotype was determined to be encoded by a polypeptide species (HpmA) with an estimated molecular size of 140 kilodaltons based on minicell polypeptide analysis of pWPM100 and its mutant derivatives. Southern blotting analysis with a HpmA-specific DNA probe revealed that this novel determinant is commonly found in both Proteus species but is not present in hemolytic isolates of M. morganii, E. coli, Citrobacter freundii, and Serratia marcescens.
Antigens, Bacterial↗
Rapid lysis technique for mycobacterial species.
The lysis of mycobacterial cells typically has been difficult and time-consuming. We report a method for the physical rupture of Mycobacterium paratuberculosis and several other members of the family Mycobacteriaceae using a Mini-Beadbeater cell disrupter (Biospec Products; Bartlesville, Okla.) and zirconium beads, a process which yields DNA and RNA of high molecular weight and in greater quantity than that obtained by rupture in a Ribi pressure cell.
Animals↗
Clinical interpretation of ultrasound measurements in preterm pregnancies with premature rupture of the membranes.
Interpreting ultrasound measurements in preterm pregnancies complicated by premature rupture of the membranes is problematic; the effects of physical compression and diminished fetal growth are unclear. To delineate these factors, we analyzed ultrasound scans from 397 preterm pregnancies, including 26 with premature rupture of the membranes. Mean biparietal diameter (BPD), abdominal circumference, femur length, and estimated fetal weight were all significantly smaller with premature rupture of the membranes than without. However, this reflected younger gestational age and diminished fetal growth, rather than physical compression. The reduction in abdominal circumference was greater than that in other indices, which further suggests compromised intrauterine growth. Clinical interpretation of ultrasound measurements should consider the increased likelihood of growth retardation with preterm premature rupture of the membranes to avoid underestimating gestational age and/or missing the diagnosis of intrauterine growth retardation.
Embryonic and Fetal Development↗
Limitations of using maximum vertical pocket and other sonographic evaluations of amniotic fluid volume to predict fetal growth: technical or physiologic?
Maximum vertical pocket measurement and subjective sonographic impression of amniotic fluid volume were studied in 487 selected pregnancies. Opiate abuse, suspected fetal growth retardation, and suspected postterm gestation were negatively correlated with maximum vertical pocket; suspected fetal growth acceleration and increasing birth weight were positively correlated. Maximum vertical pocket appeared to peak at 31 to 33 weeks (p less than 0.05), and was related to fetal growth (p less than 0.001). Subjective sonographic evaluation of amniotic fluid volume yielded comparable results. Unfortunately, neither technique could be used to accurately predict the birth of a small, appropriate, or large for gestational age infant in the individual pregnancy. Abnormal fetal growth could be reliably excluded in only a small number of cases. This and previous studies suggest that diagnosis of abnormal fetal growth by means of sonographic amniotic fluid volume evaluation could be limited by physiologic variation rather than technical error in measurement.
Amniotic Fluid↗
Employment of diabetics in a post office region.
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Absenteeism↗
Reconsideration of head compression and intraventricular hemorrhage in the vertex very-low-birth-weight fetus.
The relationship between fetal head compression and the occurrence of intraventricular hemorrhage was examined in 101 very-low-birth-weight (500 to 1500 g) vertex neonates. Peripartum events related to head compression, including parity, rupture of membranes, labor, episiotomy, use of forceps, and cesarean birth were not significantly related to intraventricular hemorrhage. A birth weight of less than 1250 g, neonatal depression (one minute Apgar score less than 7), respiratory distress syndrome, and neonatal mortality were significantly associated with intraventricular hemorrhage (P less than .05). These findings suggest that head compression in the vertex very-low-birth-weight fetus, as encountered in current obstetric practice, is not a major determinant of intraventricular hemorrhage. When premature delivery is inevitable, focusing on efforts to avoid a depressed one-minute Apgar score appears to be a more promising approach for the prevention of intraventricular hemorrhage than routinely using cesarean birth.
Adolescent↗
Fractured ureteral catheters in gynecologic oncology.
Two cases of fractured polyethylene double-pigtail ureteral catheters in pelvic cancer patients are reported. Current literature leaves a large void in guiding the management of these catheters. Surveillance techniques to help avoid complications in patients requiring these catheters are suggested.
Carcinoma, Squamous Cell↗
Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide.
A 110-kilodalton polypeptide isolated from cell-free culture supernatants of hemolytic Escherichia coli was shown to be associated with hemolytic activity. The relative amount of the extracellular 110-kilodalton species detected directly reflects the extracellular hemolysin activity associated with Escherichia coli strains harboring different hemolysin recombinant plasmids. The predicted molecular mass of the hemolysin structural gene (hlyA) based on DNA sequence analysis was 109,858 daltons. Amino-terminal amino acid sequence analysis of the 110-kilodalton polypeptide provided direct evidence that it was encoded by hlyA. Based on this information, it was also demonstrated that the HlyA polypeptide was released extracellularly without signal peptidase-like cleavage. An examination of hemolysin-specific polypeptides detected by use of recombinant plasmids in a minicell-producing strain of Escherichia coli was performed. These studies demonstrated how hemolysin-associated 110- and 58-kilodalton polypeptides detected in the minicell background could be misinterpreted as a precursor-product relationship.
Amino Acid Sequence↗
Nucleotide sequence of an Escherichia coli chromosomal hemolysin.
We determined the DNA sequence of an 8,211-base-pair region encompassing the chromosomal hemolysin, molecularly cloned from an O4 serotype strain of Escherichia coli. All four hemolysin cistrons (transcriptional order, C, A, B, and D) were encoded on the same DNA strand, and their predicted molecular masses were, respectively, 19.7, 109.8, 79.9, and 54.6 kilodaltons. The identification of pSF4000-encoded polypeptides in E. coli minicells corroborated the assignment of the predicted polypeptides for hlyC, hlyA, and hlyD. However, based on the minicell results, two polypeptides appeared to be encoded on the hlyB region, one similar in size to the predicted molecular mass of 79.9 kilodaltons, and the other a smaller 46-kilodalton polypeptide. The four hemolysin gene displayed similar codon usage, which is atypical for E. coli. This reflects the low guanine-plus-cytosine content (40.2%) of the hemolysin DNA sequence and suggests the non-E. coli origin of the hemolysin determinant. In vitro-derived deletions of the hemolysin recombinant plasmid pSF4000 indicated that a region between 433 and 301 base pairs upstream of the putative start of hlyC is necessary for hemolysin synthesis. Based on the DNA sequence, a stem-loop transcription terminator-like structure (a 16-base-pair stem followed by seven uridylates) in the mRNA was predicted distal to the C-terminal end of hlyA. A model for the general transcriptional organization of the E. coli hemolysin determinant is presented.
Amino Acid Sequence↗
Failure of exogenous progestogens to block dexamethasone-induced prostaglandin F release from the uterus of the late-pregnant cow.
The effect of exogenous progesterone or megestrol acetate (MGA) on cervical extensibility and on the concentrations of prostaglandin (PG) F in utero-ovarian plasma and progesterone, estrone sulphate and unconjugated estradiol-17 beta concentrations in jugular plasma was studied over Days 238-242 in pregnant cows given intrafetal infusions of dexamethasone to induce premature delivery. All 11 cows used in the study were given continuous intrafetal infusions of dexamethasone at a rate of 3.3 mg/day to induce premature labor. Progesterone was infused continuously at a rate of 400 mg/day for 10 day into the jugular vein of 3 of 11 cows while another 3 were given MGA at a rate of 20 mg/day. In the cows given dexamethasone alone premature parturition was induced in 2.5-4.5 days and all calves were born alive and well. However, in the progestogen-treated cows, dexamethasone failed to induce premature delivery. The cervix of these cows remained firm and unyielding during the time of the progesterone infusion. Following the withdrawal of exogenous progestogens the cervix became soft and compliant and the cows delivered within 0.5-1.5 days, but only 2 of the 6 calves were alive at birth. Hormone changes in the cows given intrafetal infusions of dexamethasone included a marked rise in utero-ovarian PGF and a concomitant decline in jugular progesterone concentrations. Plasma concentrations of estrone sulphate and estradiol-17 beta showed a small increase before delivery. An increase in utero-ovarian PGF concentrations was also observed in all of the progestogen-treated cows, although there was considerable individual variation in the timing of the PGF surge. The plasma concentrations of estrone sulphate and unconjugated estradiol-17 beta levels in the progestogen-treated cows showed the same general trend, with the levels increasing within 1-2 days of starting the dexamethasone infusion and then falling before delivery.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗