Anesthesia for non-obstetric surgery during pregnancy in a tertiary referral center: a 16-year retrospective, matched case-control, cohort study.

INTRODUCTION: This retrospective, matched case-control cohort study describes the incidence, indications, anesthesia techniques and outcomes of pregnancies complicated by surgery in a single tertiary-referral hospital. METHODS: Retrospective review of the hospital records of 171 patients who had non-obstetric surgery in the current pregnancy, between 2001 and 2016. Pregnancy outcomes of these women were firstly compared with all contemporary non-exposed patients (n=35 411), and secondly with 684 non-exposed control patients, matched for age, time of delivery and parity. RESULTS: The incidence of non-obstetric surgery during pregnancy was 0.48%, mostly performed during the second trimester (44%) and under general anesthesia (81%). Intra-abdominal surgery (44%) was the most commonly performed procedure, predominantly using laparoscopy (79%). Women undergoing surgery delivered earlier and more frequently preterm (25% vs. 17%, P=0.018); and birth weight was significantly lower [median (95% CI) 3.16 (3.06 to 3.26) vs. 3.27 (3.22 to 3.32) kg, P=0.044]. When surgery was performed under general anesthesia, low birth weight was more frequent (22% vs 6%, P=0.046). Overall pregnancy outcomes were neither influenced by trimester nor location (intra- vs extra-abdominal) of surgery. However, preterm birth rate secondary to surgery was higher for interventions during the third trimester, compared with other trimesters (10% vs 0, P <0.001). CONCLUSION: Pregnant women who underwent surgery delivered preterm more frequently and their babies had lower birth weights. Laparoscopic surgery did not increase the incidence of adverse pregnancy outcomes. General anesthesia was associated with low birth weight. Whether these associations suggest causation or reflect the severity of the underlying condition remains speculative.

Colocalization of cerebral iron with Amyloid beta in Mild Cognitive Impairment.

Quantitative Susceptibility Mapping (QSM) MRI at 7 Tesla and 11-Carbon Pittsburgh-Compound-B PET were used for investigating the relationship between brain iron and Amyloid beta (Aß) plaque-load in a context of increased risk for Alzheimer's disease (AD), as reflected by the Apolipoprotein E ε4 (APOE-e4) allele and mild cognitive impairment (MCI) in elderly subjects. Carriers of APOE-e4 with normal cognition had higher cortical Aß-plaque-load than non-carriers. In MCI an association between APOE-e4 and higher Aß-plaque-load was observable both for cortical and subcortical brain-regions. APOE-e4 and MCI was also associated with higher cortical iron. Moreover, cerebral iron significantly affected functional coupling, and was furthermore associated with increased Aß-plaque-load (R2-adjusted = 0.80, p < 0.001) and APOE-e4 carrier status (p < 0.001) in MCI. This study confirms earlier reports on an association between increased brain iron-burden and risk for neurocognitive dysfunction due to AD, and indicates that disease-progression is conferred by spatial colocalization of brain iron deposits with Aß-plaques.

Adolescent slipped capital femoral epiphysis treated by a modified Dunn osteotomy with surgical hip dislocation.

Between June 2001 and November 2008 a modified Dunn osteotomy with a surgical hip dislocation was performed in 30 hips in 28 patients with slipped capital femoral epiphysis. Complications and clinical and radiological outcomes after a mean follow-up of 3.8 years (1.0 to 8.5) were documented. Subjective outcome was assessed using the Harris hip score and the Western Ontario and McMaster Universities osteoarthritis index questionnaire. Anatomical or near-anatomical reduction was achieved in all cases. The epiphysis in one hip showed no perfusion intra-operatively and developed avascular necrosis. There was an excellent outcome in 28 hips. Failure of the implants with a need for revision surgery occurred in four hips. Anatomical reduction can be achieved by this technique, with a low risk of avascular necrosis. Cautious follow-up is necessary in order to avoid implant failure.

Anesthesia for non-obstetric surgery in the pregnant patient.

Surgery during pregnancy is relatively common. The present review of the literature will focus on relevant issues such as maternal safety during non-obstetric surgery in pregnancy, teratogenicity of anesthetic drugs, the avoidance of fetal asphyxia, the prevention of preterm labor, the safety of laparoscopy, the need to monitor the fetal heart rate and will finally give a practical approach to manage these patients.

Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex.

Editing of apolipoprotein B (apoB) mRNA requires the catalytic component APOBEC-1 together with "auxiliary" proteins that have not been conclusively characterized so far. Here we report the purification of these additional components of the apoB mRNA editing enzyme-complex from rat liver and the cDNA cloning of the novel APOBEC-1-stimulating protein (ASP). Two proteins copurified into the final active fraction and were characterized by peptide sequencing and mass spectrometry: KSRP, a 75-kDa protein originally described as a splicing regulating factor, and ASP, a hitherto unknown 65-kDa protein. Separation of these two proteins resulted in a reduction of APOBEC-1-stimulating activity. ASP represents a novel type of RNA-binding protein and contains three single-stranded RNA-binding domains in the amino-terminal half and a putative double-stranded RNA-binding domain at the carboxyl terminus. Purified recombinant glutathione S-transferase (GST)-ASP, but not recombinant GST-KSRP, stimulated recombinant GST-APOBEC-1 to edit apoB RNA in vitro. These data demonstrate that ASP is the second essential component of the apoB mRNA editing enzyme-complex. In rat liver, ASP is apparently associated with KSRP, which may confer stability to the editing enzyme-complex with its substrate apoB RNA serving as an additional auxiliary component.

Molecular heterogeneity of free PSA in sera of patients with benign and malignant prostate tumors.

OBJECTIVE: To analyze free prostate-specific antigen (f-PSA) in sera from patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to detect possible differences in subtypes as potential diagnostic parameters. MATERIALS AND METHODS: PSA was purified from sera by an immunoaffinity procedure developed on the basis of oriented antibody immobilization, and subjected to size exclusion chromatography (SEC), Western blotting, and N-terminal amino acid sequencing. RESULTS: The novel procedure allowed the purification of PSA with high yield from sera containing PSA <10 ng/ml. SEC under nonreducing conditions as well as Western blots demonstrated the presence of several molecular forms of f-PSA. Three of the smaller polypeptides exhibited the N-terminal sequence of PSA while one represented the C-terminal fragment Lys(146)-Pro(237). Shortening of some polypeptides by the N-terminal amino acid Ile(1) suggestive of aminopeptidase action was also observed. No propeptide sequence could be detected, and none of the bands from patient sera reacted with antibodies raised against propeptide antigens. BPH sera expressed higher proportions of smaller PSA fragments per unit p33, and contained significant amounts of fragments <14,000 which appeared to be very low or absent from most PCa sera. CONCLUSIONS: f-PSA as obtained from BPH and PCa sera represents a heterogeneous fraction. The major component (p33) is not in the nicked form and does not contain proPSA. Diagnostic potential could arise from the quantitative differences of the smaller PSA derivatives seen between PCa and BPH sera.

Neurotractin, a novel neurite outgrowth-promoting Ig-like protein that interacts with CEPU-1 and LAMP.

The formation of axon tracts in nervous system histogenesis is the result of selective axon fasciculation and specific growth cone guidance in embryonic development. One group of proteins implicated in neurite outgrowth, fasciculation, and guidance is the neural members of the Ig superfamily (IgSF). In an attempt to identify and characterize new proteins of this superfamily in the developing nervous system, we used a PCR-based strategy with degenerated primers that represent conserved sequences around the characteristic cysteine residues of Ig-like domains. Using this approach, we identified a novel neural IgSF member, termed neurotractin. This GPI-linked cell surface glycoprotein is composed of three Ig-like domains and belongs to the IgLON subgroup of neural IgSF members. It is expressed in two isoforms with apparent molecular masses of 50 and 37 kD, termed L-form and S-form, respectively. Monoclonal antibodies were used to analyze its biochemical features and histological distribution. Neurotractin is restricted to subsets of developing commissural and longitudinal axon tracts in the chick central nervous system. Recombinant neurotractin promotes neurite outgrowth of telencephalic neurons and interacts with the IgSF members CEPU-1 (KD = 3 x 10(-8) M) and LAMP. Our data suggest that neurotractin participates in the regulation of neurite outgrowth in the developing brain.

Identification of the integral membrane protein RM3/1 on human monocytes as a glucocorticoid-inducible member of the scavenger receptor cysteine-rich family (CD163).

The RM3/1 Ag is a membrane glycoprotein restricted to human monocytes and macrophages that evolve in the late phase of inflammation. Peptide sequence analysis of the RM3/1 protein revealed similarity to CD163, a member of the scavenger receptor cysteine-rich family. Using specific Abs (RM3/1, Ki-M8), we demonstrate an identical cellular regulation for the RM3/1 and the CD163 protein. Most notably, we show for the first time that CD163 is significantly up-regulated by glucocorticoids. In contrast, the protein is down-regulated by the immunosuppressant cyclosporin A and by phorbol esters, while the inflammatory mediator LPS has no significant influence on the expression. We describe the first isolation of a full-length cDNA of CD163 and expression of the corresponding protein. Several splice variants of CD163 exist, and we elucidated the kinetics of induction of three major mRNA splice variants by fluticasone propionate; another splice variant was proved to be unresponsive to this glucocorticoid. Taken together with a previous result showing an involvement of RM3/1 in adhesion of monocytes to the activated endothelium, we discuss that CD163 might play an important role in inflammatory processes.

Elucidation of a new biological function of an old protein: unique structure of the cobra serum albumin controls its specific toxin binding activity.

Although few proteins have been studies as thoroughly as serum albumin, a new biological property of this evolutionary ancient protein was recently discovered: The ability of cobra serum albumin (CSA) to specifically sequester lethal endogenous toxins. A study of the structural basis of this property is reported in this contribution. Two independent approaches were used to alter the structure of the CSA at defined positions: Directed mutagenesis and limited proteolysis. The conserved pattern of the disulfide linkages in the primary structure of the serum albumins showed in the case of the cobra snake (Naja naja kaouthia) an anomaly at C11 and C502, which suggested the existence of a unique spatial structure in this protein. The two cysteine residues were singly replaced with the consensus residue, i.e. C11-->F and C502-->T. The former substitution increased the specific neurotoxin binding capacity of the CSA by the factor 1.7 +/- 0.2, whereas the latter replacement reduced it to (25 +/- 2)%. The limited proteolysis yielded the large tryptic peptides T60, T40, T30 and T18, which after isolation by PAGE followed by HPLC had retained a strong toxin affinity. The location of these peptides in the amino-acid sequence was identified by Edman degradation and suggested the order of their release. On the basis of these data, a model of the unfolding and of the activity changes of the CSA caused by the structural perturbations was composed and the kinetic parameters associated with the process were evaluated. The results support the hypothesis of the existence of a structure of multiple homologous domains with a disulfide linkage between C11 and C502 in the native CSA that joins the chain ends to form a dense conformation.

Amino acid sequence of the D-galactose binding lectin II from the sponge Axinella polypoides (Schmidt) and identification of the carbohydrate binding site in lectin II and related lectin I.

The sponge Axinella polypoides contains several D-galactose binding lectins. One of the main components, lectin I was sequenced earlier, the complete sequence of the other major constituent of saline extracts, lectin II has been determined by amino acid sequencing and mass spectrometry. Both lectins have a homology of 65% to each other and both possess a disulfide loop between positions 4 and 46. As long as this loop is closed in both lectins, they can be boiled in the presence of SDS or treated with 6 mol guanidine hydrochloride without losing their hemagglutinating activity. Incubation with beta-mercaptoethanol alone does not effect the carbohydrate binding capacity either. However, reduction of the disulfide bond under chaotropic conditions destroys the activity irreversibly. This disulfide loop is also an immunologically dominant epitope in both lectins, as was revealed with monospecific polyclonal antisera. Thus, sponge lectins seem to be of different origins, since three completely different structures were described: the structure of Geodia cydonium, related to the mammalian S-type lectins with one SH-group, the Axinella lectins with one disulfide loop and the Aaptos lectins I and II with 11 cysteine residues/subunit.

Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli.

The streptococcal erythrogenic exotoxin A (SPEA) belongs to the family of bacterial superantigens and has been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Concerning its biological activity, mainly T-cell-stimulatory properties, conflicting data exist. In this study, we show that most of the SPEA preparations used so far contain biologically active contaminations. Natural SPEA from the culture supernatant of Streptococcus pyogenes NY-5 and recombinant SPEA purified from the culture filtrate of S. sanguis are strongly contaminated with DNases. We show that natural SPEA induces more tumor necrosis factor alpha (TNF-alpha) than recombinant SPEA, but we also show that DNases are able to induce TNF-alpha. In commercial SPEA preparations, we identified a highly active protease, which was shown not to be SPEB. To exclude these contaminations, we overexpressed SPEA cloned in the effective high-level expression vector pIN-III-ompA2 in Escherichia coli. The expressed SPEA shows the same amino acid composition as natural SPEA, whereas functional studies reported so far were carried out with toxins containing an incorrect amino terminus. We describe the rapid purification of lipopolysaccharide-, DNase-, and protease-free SPEA in two steps from the host's periplasm and its structural characterization by circular dichroism. Our results represent for the first time the production in E. coli of recombinant SPEA with the authentic N-terminal sequence and a proven superantigenic activity. Collectively, our results indicate that immunological studies of superantigens require highly purified substances free of biologically active contaminations.

p100: a novel proliferation-associated nuclear protein specifically restricted to cell cycle phases S, G2, and M.

By immunization with nuclear lysates of L428 cells, we raised a monoclonal mouse antibody, Ki-S2 (IgG1). In Western blots, this antibody recognizes a nuclear antigen with an apparent molecular mass of 100 kD, termed p100. Protein sequencing of p100 showed that this is a hitherto unknown protein. Immunohistochemical examination of cryostat and paraffin sections of nearly all human tissue types and neoplasms showed that p100 was exclusively expressed in the nuclei of a fraction of proliferating cells. Cell sorting and fluorescence-activated cell sorting analysis of stimulated peripheral blood mononuclear cells showed that p100 was exclusively expressed in proliferating cells from the transition G1/S until the end of cytokinesis. During mitosis, this protein is strictly associated with the spindle pole and with the mitotic spindle, whereas during S and G2, p100 is diffusely distributed throughout the cell nucleus. Immediately after completion of cytokinesis, p100 was rapidly degraded. In L428 cells, p100 is phosphorylated at least during mitosis. It has a turnover time of about 1 hour. Studies on routinely processed paraffin sections of specimens of malignant lymphoma, benign and malignant nevocellular tumors, and breast cancer showed that in all cases less than 40% of the Ki-67-positive growth fraction expressed p100. Thus, p100 might prove to be a more reliable measure of cellular proliferation and one that is more closely correlated to cancer prognosis, beyond its general biologic relevance as a cell cycle protein.

Variant-sequence transthyretin (isoleucine 122) in late-onset cardiac amyloidosis in black Americans.

BACKGROUND: After the age of 60, isolated cardiac amyloidosis is four times more common among blacks than whites in the United States; 3.9 percent of blacks are heterozygous for an amyloidogenic allele of the normal serum carrier protein transthyretin in which isoleucine is substituted for valine at position 122 (Ile 122). We hypothesized that the high prevalence of transthyretin Ile 122 is at least partially responsible for the increased frequency of senile cardiac amyloidosis among blacks. METHODS: Paraffin blocks of cardiac tissue were obtained from an earlier study of 52,370 autopsies in Los Angeles and were examined by immunohistochemical and DNA analyses. Samples were available from 32 of 55 blacks and 20 of 78 whites over 60 years of age with isolated cardiac amyloidosis and from two control groups (228 cases). RESULTS: Transthyretin amyloidosis was identified in 31 of the 32 cardiac-tissue samples from the black patients and in 19 of the 20 samples from the white patients. Six of the 26 analyzable DNA samples (23 percent) from the black patients and none of the 19 samples from the white patients were heterozygous for the Ile 122 variant. Four of 125 DNA samples obtained at autopsy (3.2 percent) from a second, more recent, age-matched cohort of blacks without amyloidosis at the same institution were heterozygous for the transthyretin Ile 122 allele. On reexamination the cardiac tissue from these four patients contained small amounts of amyloid not detected at the initial autopsies. All subjects with the Ile 122 variant had ventricular amyloid. CONCLUSIONS: The assessment of elderly black patients with unexplained heart disease should include a consideration of transthyretin amyloidosis, particularly that related to the Ile 122 allele.

Detection of human topoisomerase II alpha in cell lines and tissues: characterization of five novel monoclonal antibodies.

We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the alpha-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase II beta was ruled out by testing the antibodies on crude extracts from yeast cells expressing the beta-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology.

Cytokinin affects nuclear- and plastome-encoded energy-converting plastid enzymes.

Cytokinins induce two specific morphological alterations in mosses: (i) the differentiation of a tip-growing cell into a three-faced apical cell (the so-called bud), and (ii) the division of chloroplasts. In a developmental mutant of the moss Physcomitrella patens (Hedw.) B.S.G. (mutant PC22) impeded in both cellular differentiation (bud production) and chloroplast division, addition of cytokinin (N6-delta 2-isopentenyladenine) led to bud production after 3 d in the wild type and after 7 d in the mutant. Hormone induced a division of the mutant macrochloroplasts starting within 24 h and ongoing for 72 h. During this period the abundances of several plastid proteins changed in both genotypes as judged by two-dimensional-protein gel electrophoresis, silver staining and subsequent quantification with novel computer software. Eight of these polypeptides were isolated independently, subjected to microsequencing and thus identified, resulting in the first protein sequence data from a moss. Three polypeptides (24 kDa, 22 kDa, 20 kDa) were found to be homologous to enhancer protein OEE2 of the oxygen-evolving complex, four to represent isoforms of phosphoglycerate kinase (EC 2.7.2.3), and one was identified as the beta-chain of chloroplast ATPase (EC 3.6.1.34). Possible involvement of these key enzymes of the chloroplast energy-conversion machinery in organelle division and in cellular differentiation is discussed. Further sequence information was obtained from both subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Amounts of these polypeptides were not appreciably affected by cytokinin in moss chloroplasts.

Characterization, mapping and partial cDNA sequence of the 57-kD intracellular Ki-1 antigen.

A novel antigen was identified by the cross-reactivity of the anti-CD30 antibody Ki-1. This 57-kD intracellular Ki-1 antigen (Ki-1/57) is induced upon activation of leukocytes and is transported to the nuclear compartment. We describe the partial cloning and sequencing of the Ki-1/57 cDNA from a lambda gt 11-cDNA library derived from the Hodgkin-analogous cell line L540. New monoclonal antibodies were produced against the recombinant Ki-1/57 protein fragment which were used to confirm that the Ki-1/57 antigen is associated with kinase activity and is expressed in a variety of tumor cell lines and in activated but not resting leukocytes. The Ki-1/57 gene was mapped to the bands 9q22.3-31 of human chromosome 9. This is an area which appears to be associated with secondary chromosomal aberrations in acute myeloid leukemias.

UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli.

Steryl glucosides are characteristic lipids of plant membranes. The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067-1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.). Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments. Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively. The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide. The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity.

Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the 'Ki-67' proteins.

The monoclonal antibody (mab) Ki-67 has been used for about 10 years, mainly in tissue sections, to monitor proliferating cells, but so far only very little is known about the proteins it recognizes. The new mabs Ki-S3 and Ki-S5 detect proliferating cells in frozen and paraffin-embedded tissues. They recognize proteins with the same molecular mass as Ki-67 in Western blot and for the first time also in immunoprecipitation experiments. With these mabs we were able to enrich and purify the Ki-67 proteins. Protein sequencing of four peptides of the digested proteins corresponded to the cDNA-deduced amino acid sequence already published for the Ki-67 proteins. Since we were able to immunoprecipitate the Ki-67 proteins, we performed various immunoprecipitation experiments to obtain more information about the nature of these proteins. After radiolabelling L428 cells with [35S]-methionine we were able to immunoprecipitate the Ki-67 proteins after only 5 min of labelling time. In turnover experiments the Ki-67 proteins could not be detected 3 h after the end of labelling. These data indicate a half-life of the Ki-67 proteins of about 90 min. Labelling experiments with [32P]-orthophosphate revealed that the Ki-67 proteins are phosphorylated. After dephosphorylation was blocked with okadaic acid or cell growth was arrested by means of Colcemid, the phosphorylation of the Ki-67 proteins was greatly increased, indicating that the Ki-67 proteins are phosphorylated via serine and threonine, and that the phosphorylation of the Ki-67 proteins increases in cycling cells. Labelling experiments with [3H]-mannose and [3H]-glucose revealed that the protein is weakly N-glycosylated.

Isolation of the myc transcription factor nucleoside diphosphate kinase and the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase by cAMP affinity chromatography.

Cyclic AMP affinity chromatography applied to various mammalian tissue extracts yielded two proteins in addition to the regulatory subunits of protein kinase. This paper characterizes these proteins and provides a simple procedure for their preparation. The polypeptides (36 kDa and a 19 kDa/21 kDa doublet) were isolated from the cAMP matrix by sequential elution with cAMP solutions of increasing concentrations. Microsequencing was accomplished following chemical or enzymic degradation of isolated polypeptides. Partial amino acid sequences of the 36 kDa protein and analyses of its enzymic activity indicated identity with glyceraldehyde-3-phosphate dehydrogenase whilst the lower MW protein proved to be identical with mammalian nucleoside diphosphate kinase subunits. In both cases, binding to cAMP appeared to occur at the nucleotide (NAD and ATP, respectively) sites. In conclusion, we present a one step-procedure, applicable to tissue and cell extracts, which allows the simultaneous isolation of both glyceraldehyde-3-phosphate dehydrogenase and nucleoside diphosphate kinase. This procedure may help to elucidate the multiple functions of these two important enzymes.