Pregnancy History Influences the Level of Autophagy in Peripheral Blood Mononuclear Cells From Pregnant Women.

OBJECTIVE: Maternal immune responses are altered during pregnancy and differ between nulliparous and multiparous women. The influence of a prior gestation on autophagy in peripheral blood mononuclear cells (PBMCs) from pregnant women has not been determined and is the subject of this investigation. METHODS: Peripheral blood mononuclear cells were isolated from 212 pregnant women and immediately lysed in the presence of protease inhibitors, and the extent of autophagy was determined by quantitation of the concentration of p62 (sequestosome-1) in the lysates by enzyme-linked immunosorbent assay (ELISA). In PBMCs, the p62 level is inversely related to the extent of autophagy. The level of the stress-inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured in the lysates by ELISA. Data were analyzed by the Spearman rank correlation, Mann-Whitney U test, or Kruskal-Wallis test, as appropriate. RESULTS: The p62 concentration in PBMCs increased (autophagy decreased) with the number of previous live ( P = .0322), preterm ( P = .0143), or term ( P = .0418) deliveries. The p62 level was lower (autophagy higher) in women with a prior spontaneous pregnancy loss but no deliveries as compared to women with their first conception ( P = .0087). The intracellular hsp70 concentration correlated with the p62 level ( P < .0001). CONCLUSION: Multiparity is associated with a reduced level of autophagy in PBMCs. Dysregulated autophagy might be one mechanism leading to spontaneous abortion in nulliparous women.

A boronic-acid-based probe for fluorescence polarization assays with penicillin binding proteins and ß-lactamases.

Penicillin binding proteins (PBPs) and ß-lactamases are involved in interactions with ß-lactam antibiotics connected with both antibacterial activity and mediation of bacterial ß-lactam resistance. Current methods for identifying inhibitors of PBPs and ß-lactamases can be inefficient and are often not suitable for studying weakly and/or reversibly binding compounds. Therefore, improved ligand binding assays for PBPs and ß-lactamases are needed. We report the development of a fluorescence polarization (FP) assay for PBPs and "serine" ß-lactamases using a boronic-acid-based, reversibly binding "tracer." The tracer was designed based on a crystal structure of a covalent complex between a boronic acid and PBP1b from Streptococcus pneumoniae. The tracer bound to three different PBPs with modest affinity (K(d)=4-12 µM) and more tightly to the TEM1 serine ß-lactamase (K(d)=109 nM). ß-Lactams and other boronic acids were able to displace the tracer in competition assays. These results indicate that fluorescent boronic acids are suited to serve as reversibly binding tracers in FP-based assays with PBPs and ß-lactamases and potentially with other related enzymes.

Thrombotic thrombocytopenic purpura with complete molar pregnancy: a case report.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is extremely rare. This disease and its prompt diagnosis are important because TTP in pregnancy carries a 90% mortality rate. CASE: A 21-year-old woman underwent suction dilation and curettage for molar pregnancy. Postoperatively the patient developed severe hypertension, microangiopathic anemia, thrombocytopenia and chest pain associated with ischemic cardiac changes. Despite blood and plasma transfusions and steroid therapy, the patient continued to have worsening hemolysis and thrombocytopenia. TTP was diagnosed, and plasmapheresis led to a rapid recovery. CONCLUSION: TTP can occur with molar pregnancy. Making this diagnosis in a timely manner is crucial to ensure that potentially life-saving plasmapheresis is initiated in a timely manner. To our knowledge, this is the first reported case of thrombotic thrombocytopenic purpura with molar pregnancy.

Effects of shoulder dystocia training on the incidence of brachial plexus injury.

OBJECTIVE: We sought to determine whether implementation of shoulder dystocia training reduces the incidence of obstetric brachial plexus injury (OBPI). STUDY DESIGN: After implementing training for maternity staff, the incidence of OBPI was compared between pretraining and posttraining periods using both univariate and multivariate analyses in deliveries complicated by shoulder dystocia. RESULTS: The overall incidence of OBPI in vaginal deliveries decreased from 0.40% pretraining to 0.14% posttraining (P < .01). OBPI after shoulder dystocia dropped from 30% to 10.67% posttraining (P < .01). Maternal body mass index (P < .01) and neonatal weight (P = .02) decreased and head-to-body delivery interval increased in the posttraining period (P = .03). Only shoulder dystocia training remained associated with reduced OBPI (P = .02) after logistic regression analysis. OBPI remained less in the posttraining period (P = .01), even after excluding all neonates with birthweights >2 SD above the mean. CONCLUSION: Shoulder dystocia training was associated with a lower incidence of OBPI and the incidence of OBPI in births complicated by shoulder dystocia.

Structure guided development of potent reversibly binding penicillin binding protein inhibitors.

Following from the evaluation of different types of electrophiles, combined modeling and crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding protein. The results suggest that a structurally informed approach to penicillin binding protein inhibition will be useful for the development of both improved reversibly binding inhibitors, including boronic acids, and acylating inhibitors, such as ß-lactams.

Observations on the deprotection of pinanediol and pinacol boronate esters via fluorinated intermediates.

Methods for the deprotection of pinanediol and pinacol esters of various boronic acids via fluoroborane intermediates were evaluated. Treatment of the boronate esters with potassium hydrogen difluoride normally gives trifluoroborate salts; in the case of alpha-amido alkyl or o-amido phenyl boronate esters, aqueous workup gives difluoroboranes. Procedures for transformation of both trifluoroborates and difluoroboranes to free boronic acids are described.

Synthesis and evaluation of 3-(dihydroxyboryl)benzoic acids as D,D-carboxypeptidase R39 inhibitors.

Penicillin binding proteins (PBPs) catalyze steps in the biosynthesis of bacterial cell walls and are the targets for the beta-lactam antibiotics. Non-beta-lactam based antibiotics that target PBPs are of interest because bacteria have evolved resistance to the beta-lactam antibiotics. Boronic acids have been developed as inhibitors of the mechanistically related serine beta-lactamases and serine proteases; however, they have not been explored extensively as PBP inhibitors. Here we report aromatic boronic acid inhibitors of the D,D-carboxypeptidase R39 from Actinomadura sp. strain. Analogues of an initially identified inhibitor [3-(dihydroxyboryl)benzoic acid 1, IC(50) 400 microM] were prepared via routes involving pinacol boronate esters, which were deprotected via a two-stage procedure involving intermediate trifluorborate salts that were hydrolyzed to provide the free boronic acids. 3-(Dihydroxyboryl)benzoic acid analogues containing an amide substituent in the meta, but not ortho position were up to 17-fold more potent inhibitors of the R39 PBP and displayed some activity against other PBPs. These compounds may be useful for the development of even more potent boronic acid based PBP inhibitors with a broad spectrum of antibacterial activity.

A solid-state NMR study of the interaction of fish antifreeze proteins with phospholipid membranes.

Fish antifreeze proteins and glycoproteins (AF(G)Ps) prevent ice crystal growth and are able to protect mammalian cells and tissues from hypothermic damage in the sub-zero Polar oceans. This protective mechanism is not fully understood, and further data is required to explain how AF(G)Ps are able to stabilize lipid membranes as they pass through their phase transition temperatures. Solid-state NMR spectroscopy was used as a direct method to study the interaction of the 37-residue alpha-helical type I AFP, TTTT, and the low molecular weight fraction glycoprotein, AFGP8, with dimyristoylphosphatidylcholine membranes above and below the gel-fluid phase transition temperature. In contrast to previous studies in fluid phase bilayers these experiments have provided direct information regarding both the mobility of the phosphate headgroups and perturbation of the acyl chains at a range of temperatures under identical conditions on the same sample. At 5 degrees C changes in the 2H and 31P spectra and a dramatic increase in the 31P T1 relaxation times were consistent with a significant disruption of the membrane by TTTT. Heating to 30 degrees C appeared to expel the peptide from the lipid and re-cooling showed that the interaction of TTTT was not reversible. By contrast, 31P spectra of the membranes with AFGP8 were consistent with interaction with the phosphate headgroups at both 5 and 30 degrees C. Although both peptides interact with the phospholipid bilayer surface, which may stabilize the membrane at lower temperatures, the longer 31P T1 values and the 2H NMR data obtained for TTTT compared with AFGP8 suggest that TTTT causes a greater reduction of phosphate headgroup mobility and has a greater effect on the lipid acyl chains at 5 degrees C.

Applications of type I antifreeze proteins: studies with model membranes & cryoprotectant properties.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), found in the body fluids of many species of polar fish allow them to survive in waters colder than the equilibrium freezing point of their blood and other internal fluids. Despite their structural diversity, all AF(G)Ps kinetically depress the temperature at which ice grows in a non-colligative manner and hence exhibit thermal hysteresis. AF(G)Ps also share the ability to interact with and protect mammalian cells and tissues from hypothermic damage (e.g., improved storage of human blood platelets at low temperatures), and are able to stabilize or disrupt membrane composition during low temperature and freezing stress (e.g., cryoprotectant properties in stabilization of sperm and oocytes). This review will summarize studies of AFPs with phospholipids and plant lipids, proposed mechanisms for inhibition of leakage from membranes, and cryoprotectant studies with biological samples. The major focus will be on the alpha-helical type I antifreeze proteins, and synthetic mutants, that have been most widely studied. For completeness, data on glycoproteins will also be presented. While a number of models to explain stabilization and destabilization of different lipid systems have been proposed, it is currently not possible to predict whether a particular AFP will stabilize or destabilize a given lipid system. Furthermore the relationship between the antifreeze property of thermal hysteresis and membrane stabilization is unknown. This lack of detailed knowledge about how AFPs function in the presence of different types of materials has hampered progress toward the development of antifreezes for cold storage of cells, tissues, and organs.

Diffusion NMR studies on fish antifreeze proteins and synthetic analogues.

Pulsed field gradient spin echo NMR spectroscopy was used to measure diffusion coefficients of the alpha-helical type I antifreeze protein from the winter flounder, two synthetic derivatives in which the four Thr residues were replaced with Val and Ala, respectively, and the low molecular weight fraction antifreeze glycoprotein. Under the conditions studied, the natural type I antifreeze protein and low molecular weight glycoprotein gave diffusion values that were consistent with the presence of monomeric protein in solution. While significant aggregation of the Ala analogue was observed (2-10 mM), there was no evidence for aggregation in the Val analogue (1-3 mM). These results are compared with previously reported solubility and thermal hysteresis data and the implications for the design of synthetic antifreeze proteins are discussed.

Synthesis of N-benzylated-2-aminoquinolines as ligands for the Tec SH3 domain.

In recent work, we have been developing 2-aminoquinolines as ligands for Src Homology 3 (SH3) domains, so far the only reported examples of small-molecule ligands for these domains. In this paper, we report the synthesis of a series of N-benzylated-2-aminoquinolines by reductive amination of aryl aldehydes with 2-aminoquinoline. These ligands bound the SH3 domain with ca. one and a half to twofold reduced affinity relative to 2-aminoquinoline; however, some evidence was found to suggest that the benzylic substituents made new contacts with the SH3 domain surface. These results provide useful SAR information that may assist in future ligand design.

Identification and specificity studies of small-molecule ligands for SH3 protein domains.

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.