Activation of the renin-angiotensin system contributes significantly to the pathophysiology of oliguria in patients undergoing posterior spinal fusion.

BACKGROUND AND OBJECTIVE: This study was undertaken to investigate the role played by antidiuretic hormone (ADH), the renin-angiotensin system and atrial natriuretic factor (ANF) in the oliguria in patients undergoing spinal fusion. METHODS: Sixteen patients undergoing posterior spinal fusion using isoflurane and fentanyl (n = 8) or sufentanil (n = 8) had renin, aldosterone, ADH and ANF measurements. RESULTS: Compared to the non-oliguric patients, the oliguric patients had a higher number of fused vertebrae 10.5 +/- 1.3 vs. 9.0 +/- 0.5 (P = 0.01) and higher renin values at 12h (3.3 +/- 3.2 vs. 0.7 +/- 0.6 ng L(-1) s(-1), P = 0.04). Hormonal values that had a significant correlation with intraoperative diuresis were: renin at 0.5 h (r2 = 0.26, P = 0.04), aldosterone at 0.5 h (r2 = 0.30, P = 0.03) and ANF at 0.5 h (r2 = 0.32, P = 0.02). Those that had a significant correlation with the mean postoperative diuresis were: renin at 6 h (r2 = 0.29, P = 0.03), 8h (r2 = 0.26, P = 0.04) and 12h (r2 = 0.31, P = 0.03), aldosterone at 6h (r2 = 0.54, P = 0.001), 8h (r2 = 0.40, P = 0.01) and 12h (r2 = 0.32, P = 0.03), ADH at 24h (r2 = 0.38, P = 0.01) and ANF at 6h (r2 = 0.26, P = 0.045). Using stepwise regression, excluding hormonal values, only two continuous variables had a significant correlation with the mean postoperative diuresis: the number of fused vertebrae (P = 0.02) and the length of surgery (P = 0.02). CONCLUSION: Activation of the renin-angiotensin system is the major cause of the early intraoperative oliguria. ADH and the renin-angiotensin system are both involved in the pathophysiology of postoperative oliguria in patients undergoing spinal fusion.

Cardiopulmonary bypass induces significant platelet activation in children undergoing open-heart surgery.

BACKGROUND AND OBJECTIVES: To evaluate the effects of cardiopulmonary bypass (CPB) on platelet function in children undergoing open-heart surgery. METHODS: Data from 16 consecutive children undergoing cardiac surgery with CPB were prospectively collected. Blood samples of 10 mL were collected via the central venous line immediately before and after CPB for CD62 measurements by flow cytometry. RESULTS: Ten children had acyanotic heart disease (median age 3 yr, range 1.8-14) and six had a cyanotic defect (median age 4yr, range 2-14). The platelet count decreased significantly with CPB in both groups: from 163.5 (130-201) to 93.5 (57-186) x 10(3) microL(-1) in acyanotic children and from 139.5 (77-212) to 75 (43-99) x 10(3) microL(-1) in cyanotic children (P < 0.0001). The percentage of activated platelets was significantly lower in acyanotic children at baseline: 1% (0-23%) vs. 5% (3-8%) (P = 0.07). CPB increased the percentage of activated platelets significantly in both groups: post-bypass the values were 10% (range 1-17%) in acyanotic children and 7% (range 1-30%) in cyanotic children (P = 0.03). The increase in the percentage of activated platelets did not differ between the two study groups (P = 0.11). CONCLUSION: CPB induces significant platelet activation in children undergoing open-heart surgery.

Inactivation of the Streptococcus mutans fxpC gene confers resistance to xylitol, a caries-preventive natural carbohydrate sweetener.

Xylitol is transported by Streptococcus mutans via a constitutive phosphoenolpyruvate:fructose phosphotransferase system (PTS) composed of a IIABC protein. Spontaneous xylitol-resistant strains are depleted in constitutive fructose-PTS activity, exhibit additional phenotypes, and are associated with the caries-preventive properties of xylitol. Polymerase chain-reactions and chromosome walking were used to clone the fxp operon that codes for the constitutive fructose/xylitol-PTS. The operon contained three open reading frames: fxpA, which coded for a putative regulatory protein of the deoxyribose repressor (DeoR) family, fxpB, which coded for a 1-phosphofructokinase, and fxpC, which coded for a IIABC protein of the fructose-PTS family. Northern blot analysis revealed that these genes were co-transcribed into a 4.4-kb mRNA even in the absence of fructose. Inactivation of the fxpC gene conferred resistance to xylitol, confirming its function. The fxp operon is also present in the genomes of other xylitol-sensitive streptococci, which could explain their sensitivity to xylitol.

Regulation of the pts operon in low G+C Gram-positive bacteria.

The sugar transport system called phosphoenolpyruvate: sugar phosphotransferase (PTS) is widespread among eubacteria. Its is generally composed of two cytoplasmic proteins, HPr and El, which are found in all bacteria possessing a PTS, and a family of Ells whose number, specificity, and molecular structure in terms of domain arrangement vary from species to species. In low G+C Gram-positive bacteria, the genes coding for the general proteins HPr and El, designated ptsH and ptsl respectively, are organized into the pts operon. In this paper, we summarize current knowledge about the regulation of the pts operon in low G+C Gram-positive bacteria. Physiological data indicate that El and most particularly HPr make up a substantial proportion of cellular proteins. Their synthesis is not coordinated and is influenced by environmental factors. The principal DNA cis-elements involved in the regulation of pts operon transcription are a strong promoter whose sequence and structure are very similar to those of the canonical promoter recognized by the Escherichia coli and Bacillus subtilis major RNA polymerases, a 5'-untranslated region, a rho-dependent terminator located at the 5' end of ptsl, and an intrinsic terminator located downstream from ptsl. Analysis of ptsH and ptsl Shine-Dalgarno sequences as well as experimental results obtained with a Streptococcus salivarius mutant suggest that the expression of HPr and El is also controlled at the translation level.

The phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius. Functional and biochemical characterization of IIABL(Man) and IIABH(Man).

Previous studies have suggested that the phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius consists of a nonphosphorylated enzyme II domain that functions in tandem with a separate enzymatic complex called III(Man). The III(Man) complex is believed to be composed of two protein dimers with molecular masses of approximately 72 kDa. Analysis of these proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has indicated that one dimer is composed of two 38.9-kDa subunits called IIIH(Man), and the other of two 35.2-kDa subunits called IIIL(Man). This study was undertaken to determine (1) the number and nature of the phosphorylated residue(s) on IIIH(Man) and IIIL(Man) and the phosphorylation sequence allowing the transfer of the phosphoryl group from HPr(His approximately P) to the mannose:PTS substrates; (2) whether IIIH(Man) and IIIL(Man) originate from two different genes or result from a posttranslational modification; and (3) whether these two proteins are involved in the phosphorylation of 2-deoxyglucose, a substrate of the phosphoenolpyruvate:mannose phosphotransferase system. We showed that both IIIH(Man) and IIIL(Man) were phosphorylated on two histidine residues. One phosphate bond was heat-labile (phosphorylation at the N1 position of the imidazole ring), while the second was heat-resistant (phosphorylation at the N3 position of the imidazole ring). The sequence of the first phosphorylation site was deduced by comparing the N-terminal amino acid sequence of both forms of III(Man) with IIA domains of the EII-mannose family. The sequences of both forms were identical over the 15 first amino acids, that is, MIGIIIASHGKFAEG. The sequence of the second phosphorylation site was determined for IIIL(Man) as IHGQVATNxTP. Hence, IIIH(Man) and IIIL(Man) are PTS proteins of the IIAB type and should be renamed IIABH(Man) and IIABL(Man). IIABH(Man) and IIABL(Man) had different peptide profiles after digestion with proteases, indicating that these two proteins are encoded by two different genes. In vitro PEP-dependent phosphorylation assays conducted with a spontaneous mutant devoid of both forms of IIAB(Man) suggested that the phosphoenolpyruvate:mannose phosphotransferase system of S. salivarius is composed of an uncharacterized nonphosphorylated membrane component that works in tandem with IIABL(Man). The physiological functions of IIABH(Man) remain unknown.

Predicting blood loss in surgery for idiopathic scoliosis.

The authors attempted to determine the relative importance of factors that influence bleeding during and after spinal fusion. Data from 30 ASA I patients with idiopathic scoliosis were prospectively collected and analyzed. Intraoperative bleeding was 1971 +/- 831 ml (mean +/- SD) (61.5 +/- 27% of estimated blood volume (EBV) and correlated with the number of fused vertebrae (r = 0.66, P < 0.0001) and the duration of surgery (r = 0.46, P = 0.0105). There was no correlation between intraoperative bleeding and the Cobb curve angle (43 to 86 degrees), the mean arterial blood pressure (MAP) (63 to 86 mmHg), the central venous pressure (CVP), the quantity of epinephrine infiltrated, muscle relaxants or opioids used, nor in the type of opioids used, the minimal body temperature or whether stored or autologous blood was used. Postoperative bleeding was 1383 +/- 369 ml (43.1 +/- 11.7% of EBV) and correlated with the length of time the Hemovac drain was in place (r = 0.40, P = 0.0285) and MAP (r = 0.40, P = 0.0285). There was no correlation between postoperative and intraoperative bleeding nor in the number of fused vertebrae. Six patients had greater postoperative than intraoperative bleeding. The total bleeding (intra- plus postoperative) was 3347 +/- 920 ml (104.2 +/- 30.6 of EBV) and correlated with the number of fused vertebrae (r = 0.63, P = 0.0001) and with the duration of surgery (r = 0.42, P = 0.0208). We conclude that the number of fused vertebrae is the key factor in predicting intraoperative and total bleeding. Postoperative bleeding is considerable (up to 76.9% of EBV).(ABSTRACT TRUNCATED AT 250 WORDS)

IS1139 from Streptococcus salivarius: identification and characterization of an insertion sequence-like element related to mobile DNA elements from gram-negative bacteria.

An insertion sequence-like element, IS1139, was cloned and sequenced from Streptococcus salivarius ATCC 25975 chromosome. This insertion sequence-like element is 1168 bp long and is delimited by inverted repeats of 29 bp and by a duplicated sequence of 6 bp. This IS possesses an open reading frame that codes for a putative transposase of 339 amino acids which has, respectively, 94, 35, 33, and 30% amino-acid identity with the transposases of IS1161 from S. salivarius ATCC 25975, IS4351 from Bacteroides fragilis, IS30 from Escherichia coli, and IS1086 from Alcaligenes eutrophus. Sequence analysis revealed that these transposases may have evolved from a common ancestral gene. Southern hybridization of restriction endonuclease-digested genomic DNA from 21 strains of oral streptococci, using a probe specific to the transposase-encoding gene (tnpA), revealed that IS1139 is found in two strains of S. salivarius, ATCC 25975 and ATCC 13419, in eight and two copies, respectively.

Fusion of the genes encoding Escherichia coli heat-stable enterotoxin b (STb) and the maltose-binding protein to obtain mature STb enterotoxin.

The heat-stable enterotoxin b gene (estB) of Escherichia coli was fused to the gene for maltose-binding protein (malE). The estB gene was cloned into the pMAL-p vector using PCR. The construct consists of the signal sequence of maltose-binding protein, which directs the export of the fusion protein to the periplasm, and the maltose-binding protein fused to the STb polypeptide. A sequence encoding a factor Xa cleavage site is present between malE and estB. The fused genes are controlled by Ptac, a strong inducible promoter. Following IPTG induction, the recombinant strain expressed a 47 kDa protein, which was easily purified from osmotic shock fluid by using preparative electrophoresis and electroelution. Cleavage of the fusion protein with factor Xa generated the maltose-binding protein (42 kDa) and a polypeptide of approximately 5 kDa, corresponding to the molecular mass of mature STb. A monospecific polyclonal rabbit antiserum raised against purified STb reacted in immunoblot with the fusion protein and the cleaved-off peptide. A positive response was observed when testing the osmotic shock fluid containing the fusion protein in a rat intestinal loop assay. On average, 3-4 mg of MBP-STb protein was recovered per litre of induced recombinant strain.

Reduction of Selenate and Selenite to Elemental Selenium by a Pseudomonas stutzeri Isolate.

A Pseudomonas stutzeri isolate rapidly reduced both selenite and selenate ions to elemental selenium at initial concentrations of both anions of up to 48.1 mM. Optimal selenium reduction occurred under aerobic conditions between pH 7.0 and 9.0 and at temperatures of 25 to 35 degrees C. Reduction of both selenite and selenate was unaffected by a number of anions except for sulfite, chromate, and tungstate ions, which inhibited both growth and reduction.

Lidocaine hydrocarbonate and lidocaine hydrochloride for cesarean section: transplacental passage and neonatal effects.

Twenty-six patients, ASA physical status 1, scheduled for elective cesarean section, were divided at random into two groups and received via an epidural catheter 20 ml of 2.2% lidocaine hydrocarbonate (17.3 mg.ml-1 lidocaine base) with 5 micrograms.ml-1 epinephrine freshly added (Group CO2 = 13 patients) or 20 ml of 2% lidocaine hydrochloride (17.3 mg.ml-1 lidocaine base) also with 5 micrograms.ml-1 epinephrine freshly added. Following clampage of the umbilical cord (at 40.1 +/- 4.9 min after the injection of lidocaine for the CO2 group and at 41.0 +/- 5.4 min for the HCl group), serum concentrations of lidocaine were measured both in the mother and in the umbilical vein. All newborns were examined by the same blinded pediatrician with Apgar scores at 1, 5 and 10 min and with Neurobehavioral Adaptive Capacity Scores (NACS) at 15 min, 2 h and 24 h. The concentrations of lidocaine in the serum were comparable in both groups: in the mothers 8.61 +/- 1.48 mumol.l-1 for the CO2 group vs 8.04 +/- 2.36 mumol.l-1 for the HCl group and in the newborns 3.86 +/- 0.84 mumol.l-1 for the CO2 group vs 3.92 +/- 0.95 mumol.l-1 for the HCl group. The ratio of umbilical vein to maternal vein concentrations of lidocaine was also similar in both groups: 0.45 +/- 0.07 for the CO2 group vs 0.54 +/- 0.24 for the HCl group. The percentage of newborns with a normal NACS (score > or = 35/40) was equal in both groups, i.e. 91% at 15 min and 2 h of life and 100% at 24 h of life.(ABSTRACT TRUNCATED AT 250 WORDS)

A trial of desmopressin to reduce blood loss in patients undergoing spinal fusion for idiopathic scoliosis.

Desmopressin (DDAVP) has been reported to reduce bleeding in patients undergoing spinal fusion. To evaluate its efficacy in normal patients, 30 healthy young patients (ASA physical status I or II) undergoing spinal fusion for idiopathic scoliosis were randomly allocated to receive either 100 mL of physiologic saline solution (placebo group) or DDAVP (10 micrograms/m2 of body surface area) (DDAVP group) in a prospective, double-blind trial. Intraoperative blood loss was measured by weighing sponges and suction drainage and postoperative bleeding by wound drainage. The amount of blood loss expressed as a percent of the estimated blood volume was similar in both groups during the intraoperative period (67.0% +/- 28.8% [mean +/- SD] placebo group vs 57.4% +/- 26.5% DDAVP group), the postoperative period up to 24 h (32.5% +/- 6.4% placebo group vs 31.1% +/- 10.6% DDAVP group), and both periods (94.3% +/- 29.4% placebo group vs 88.2% +/- 30.7% DDAVP group). With the dose used in our study, we conclude that DDAVP does not reduce surgical bleeding in patients undergoing spinal fusion for idiopathic scoliosis.

Immunodot detection of Escherichia coli heat-stable enterotoxin b by using enhanced chemiluminescence reaction.

An indirect immunodot assay with rabbit antibodies raised against purified heat-stable enterotoxin type b (STb) and with a Western blotting (immunoblotting) detection system (ECL; Amersham International plc, Amersham, United Kingdom) was developed for the detection of STb toxin. Culture supernatants of 62 Escherichia coli isolates from pigs with diarrhea were blotted onto nitrocellulose and incubated with anti-STb serum. The chemiluminescence produced by the action of horseradish peroxidase with luminol and H2O2 was recorded by exposure of X-ray film. Over 90% correlation was observed between the rat or pig intestinal ligated loop assay and a radioactive DNA probe and the ECL immunodot assay for the detection of STb. In addition, using this new and sensitive technique, we could detect STb in the feces of a newborn pig inoculated with an STb-producing E. coli strain. Detection of STb-producing E. coli in pigs with diarrhea will be greatly facilitated by the use of this convenient and rapid diagnostic assay.

Evaluation of three new techniques for the detection of STb-positive Escherichia coli strains.

A study was conducted to compare different techniques for the detection of heat-stable enterotoxin b (STb)-positive E. coli strains. Antisera against purified STb was used to develop an enzyme-linked immunosorbent assay (ELISA). STb-positive strains identified by ELISA were tested for bioactivity in rat jejunal loops. Our ELISA was as sensitive as, but less specific than, the bioassay for detection of STb-positive strains. A non-radioactive DNA probe to detect the gene coding for STb was also developed by incorporating digoxigenin-11-dUTP into DNA by the random primed labelling technique. The non-radioactive digoxigenin-labelled DNA probe demonstrated a similar detectability to the radioactive probe and was more convenient to manipulate but was less sensitive and specific than the bioassay and the radioactive probe. In addition, the polymerase chain reaction (PCR) was used to amplify a specific portion of the gene coding for STb. The PCR was a highly specific and practical technique for the detection of STb-positive strains. All E. coli strains tested containing the STb gene produced the STb toxin.

Detection of genes for fimbrial antigens and enterotoxins associated with Escherichia coli serogroups isolated from pigs with diarrhea.

A total of 1,226 Escherichia coli strains isolated from 1979 to 1989 from pigs with diarrhea were examined for serogroup and fimbrial antigen F4 (K88) production. Four main patterns of isolation of the various serogroups were observed, depending on the ages of the pigs from which isolates were obtained and the production of F4. In pattern I, serogroups O8:K"S16", O9:K35, O9/O101:K30, O9/O101:K103, O9 (group), O20:K101, and O64:K"V142" were predominant in pigs aged 0 to 6 days (41.9% of isolates) and were less frequent in pigs aged 7 to 27 days (24.6% of isolates) but were rarely found in pigs aged 28 to 60 days (4.0% of isolates). In pattern II, the F4-associated serogroups O8:K"4627", O157:K"V17", O149:K91, and O147:K89 were predominant in pigs aged 7 to 27 days (29.8% of isolates) and in pigs aged 28 to 60 days (35.0% of isolates). In pattern III, serogroups O8 (group), O115:K"V165", and O147:K89 were rarely isolated from pigs aged 0 to 6 days but were equally distributed in pigs aged 7 to 27 days (10.1% of isolates) and in pigs aged 28 to 60 days (10.9% of isolates). In pattern IV, serogroups O138:K81, O139:K82, O141:K85ac, O45:K"E65", and O26:K60 were most frequently isolated in pigs aged 28 to 60 days (19.3% isolates). Over the period from 1979 to 1989, the proportion of isolates belonging to serogroups of pattern II and the proportion of F4 isolates within the serogroup O157:K"V17" declined, whereas the proportion of isolates of serogroups O147:K89, O8:K"S16", and O9:K35 increased. For 228 isolates selected from the most important serogroups, good agreement was observed between the results of gene probes and immunofluorescence for the detection of fimbrial antigens F4 (K88), F5 (K99), F6 (987P), and F41 and between the results of gene probes and biological assays for the detection of heat-labile enterotoxin (LT) and heat-stable enterotoxins a and b (STa and STb). The STa gene was mostly associated with isolates of pattern I serogroups, which had the F5, F6, and F41 genes alone or in various combinations. The LT and/or STb genes, with the F4 gene, mostly were observed in isolates of pattern II serogroups. The STb gene alone was observed mostly in isolates of pattern III serogroups, although isolates were negative for all fimbrial antigen genes. Similarly, isolates of pattern IV serogroups were negative for all fimbrial antigen genes and rarely positive for the enterotoxin genes. However, verotoxin production was associated with isolates of serogroups O138:K81 and O139:K82. The most important pathotypes among enterotoxigenic isolates in this study were F4:LT:STb, F5:STa, STb, F5:F41:STa, F4:STb, F6, STa, and LT.

Characterization of Escherichia coli strains producing heat-stable enterotoxin b (STb) isolated from humans with diarrhea.

Two of 49 cytolethal distending toxin-producing strains of Escherichia coli isolated from human stools contained the gene coding for heat-stable enterotoxin b (STb), as detected by a colony hybridization assay. The STb gene was found to be on a 70-kb plasmid also coding for heat-labile enterotoxin (pLT-I). Restriction endonuclease analysis showed the STb gene from human isolates to be similar to the STb gene found in porcine strains. Moreover, by enzymatic amplification based on oligonucleotide primers designed from a porcine STb sequence, the expected portion of the STb gene was amplified for the human E. coli strains. The STb enterotoxin from these strains was bioactive in rat jejunal loops and was detected with an enzyme-linked immunosorbent assay by using polyclonal antiserum raised against purified porcine STb toxin.

Long-term follow-up of patients who underwent unilateral nephrectomy in childhood.

The long-term damaging potential of remnant nephron hyperperfusion was investigated in patients who had undergone unilateral nephrectomy in childhood. 27 such patients were examined after a mean of 23.3 years postnephrectomy. The average creatinine clearance was 83.9 +/- 16.5 ml/min/1.73 m2 or 74.3% of that in healthy controls with two kidneys; it was a value similar to that reported 3 to 6 months postnephrectomy in kidney donors. Age at the time of nephrectomy, duration of follow-up, or sex had no influence on the residual creatinine clearance. None of these patients had clinically important hypertension or proteinuria. Since so little evidence of kidney damage could be documented after such a long observation period, hyperperfusion would seem to be seldom of clinical importance in man unless other factors were present.

Local renal graft irradiation in children.

Between 1974-1979, 64 renal cadaveric transplants were performed in 54 pediatric recipients at our institution. Forty eight of these 64 transplants experienced at least one episode of acute rejection. These patients were divided in two equal groups including 24 transplants in 21 recipients, one group treated with chemical immunosuppression alone, the other group treated by chemical immunosuppression and radiotherapy. Kidney survival at 2 years was 54.1% (13/24) in the control group treated by chemical immunosuppression alone. In the group treated by radiotherapy and immunosuppression, kidney survival after 2 years gave a success rate of 45.8% (11/24). Thus, it would appear that addition of radiotherapy to standard immunosuppressive treatment exerted no beneficial long term effect in acutely rejected renal transplants. In view of the disappointing results obtained with radiotherapy, it is felt that this mode of treatment should be restricted to use in particular circumstances as a temporary means of immunosuppression where systemic immunosuppression is hazardous.

Renal osteodystrophy in children treated with 1,25-dihydroxy-cholecalciferol [1,25-(OH)2D3]. Histologic bone studies.

Eleven uremic children with osteodystrophy aged 3 to 17 years were studied during administration of 1,25-(OH)2D3 for periods up to 21 months. Nine children presented with pure hyperparathyroidism, one with osteomalacia and one with mixed bone disease. Bone biopsies were performed before initiation of therapy and after 6 to 21 months of treatment following double tetracycline labeling. Skeletal lesions were improved but not cured in 5 of 9 children with hyperparathyroidism. In three instances lesions remained unchanged and worsened in one. No significant change was observed in the child with osteomalacia. Moderate improvement was noted in the patient with mixed bone disease. The propensity to develop hypercalcemia was the major factor associated with treatment failure since it precluded administration of adequate amounts of medication. Therapy with 1,25-(OH)2D3 was associated with a spectacular improvement in growth velocity in two of six children under age twelve.