Molecular dynamics study of water channels in natural and synthetic amyloid-ß fibrils.

We compared all-atom explicit solvent molecular dynamics simulations of three types of Aß(1-40) fibrils: brain-seeded fibrils (2M4J, with a threefold axial symmetry) and the other two, all-synthetic fibril polymorphs (2LMN and 2LMP, made under different fibrillization conditions). Fibril models were constructed using either a finite or an infinite number of layers made using periodic images. These studies yielded four conclusions. First, finite fibrils tend to unravel in a manner reminiscent of fibril dissolution, while infinite fibrils were more stable during simulations. Second, salt bridges in these fibrils remained stable in those fibrils that contained them initially, and those without salt bridges did not develop them over the time course of the simulations. Third, all fibrils tended to develop a "stagger" or register shift of ß-strands along the fibril axis. Fourth and most importantly, the brain-seeded, 2M4J, infinite fibrils allowed bidirectional transport of water in and out of the central longitudinal core of the fibril by rapidly developing gaps at the fibril vertices. 2LMP fibrils also showed this behavior, although to a lesser extent. The diffusion of water molecules in the fibril core region involved two dynamical states: a localized state and directed diffusion in the presence of obstacles. These observations provided support for the hypothesis that Aß fibrils could act as nanotubes. At least some Aß oligomers resembled fibrils structurally in having parallel, in-register ß-sheets and a sheet-turn-sheet motif. Thus, our findings could have implications for Aß cytotoxicity, which may occur through the ability of oligomers to form abnormal water and ion channels in cell membranes.

Ionic strength independence of charge distributions in solvation of biomolecules.

Electrostatic forces enormously impact the structure, interactions, and function of biomolecules. We perform all-atom molecular dynamics simulations for 5 proteins and 5 RNAs to determine the dependence on ionic strength of the ion and water charge distributions surrounding the biomolecules, as well as the contributions of ions to the electrostatic free energy of interaction between the biomolecule and the surrounding salt solution (for a total of 40 different biomolecule/solvent combinations). Although water provides the dominant contribution to the charge density distribution and to the electrostatic potential even in 1M NaCl solutions, the contributions of water molecules and of ions to the total electrostatic interaction free energy with the solvated biomolecule are comparable. The electrostatic biomolecule/solvent interaction energies and the total charge distribution exhibit a remarkable insensitivity to salt concentrations over a huge range of salt concentrations (20 mM to 1M NaCl). The electrostatic potentials near the biomolecule's surface obtained from the MD simulations differ markedly, as expected, from the potentials predicted by continuum dielectric models, even though the total electrostatic interaction free energies are within 11% of each other.

Gene expression of the constant region of the heavy chain of immunoglobulin G (IgG CRHC) is down-regulated in human decidua in association with preeclampsia.

An aberrant interaction at the maternal/fetal interface between the genetically distinct fetal trophoblast cells and cells of the maternal decidua has been proposed as an initiating factor in one of the major complications of human pregnancy, preeclampsia. Biochemical and epidemiological studies suggest that the immune system plays an important role in preeclampsia. Thus, the aim of this study was to determine the decidual gene expression status in preeclampsia of one of the key components of the adaptive immune system. Total RNA was extracted from decidua collected from women with normal pregnancies and those complicated by preeclampsia. Reverse Northern analysis was performed on 72 cDNAs from human decidua and differentially expressed genes identified were analysed further using semi-quantitative RT-PCR and Northern blot analysis. Expression of the gene encoding the constant region of the heavy chain of immunoglobulin G (IgG CRHC) was shown to be down-regulated in association with preeclampsia. These data support the hypothesis that immune maladaptation may play an important role in the pathogenesis of preeclampsia.

Localization of insulin-like growth factor-I mRNA in rat brain by in situ hybridization--relationship to IGF-I receptors.

Recent evidence has demonstrated regional synthesis of insulin-like growth factor I (IGF-I) in rat brain, which is also known to contain widespread specific type I IGF receptors. In order to precisely define sites of IGF-I mRNA synthesis, and their relationship to IGF-I receptor sites, we have applied the techniques of in situ hybridization and in vitro receptor autoradiography in rat brain. Frozen sections of adult rat brain and liver were hybridized with 32P-labeled cDNA inserts for human IGF-I (780 base pairs) or a positive control transthyretin cDNA (1430 base pairs) probe, or a series of negative probes, followed by film or emulsion autoradiography. Receptor autoradiography was performed on similar sections using 125I-IGF-I in buffer, some chambers containing excess unlabeled IGF-I. Hybridization of IGF-I probe was clearly seen only in three major brain regions: the olfactory bulb, hippocampus and cerebellum, whereas transthyretin only hybridized to choroid plexus as expected, and other probes showed no hybridization. In olfactory bulb, hybridization was greatest in the internal granular and mitral cell layers, with lower levels in the glomerular layer, where IGF-I receptors were concentrated. In hippocampus, hybridization was to pyramidal cells of Ammon's horn in CA1 and CA2 layers and dentate gyrus, with some labeling in CA3. IGF-I receptors were most dense in CA2, CA3, CA4, and dentate gyrus. In cerebellum, hybridization was to the granule cell layer, with IGF-I receptors primarily in the adjacent molecular layer. We have clearly demonstrated precise sites of local IGF-I synthesis in adult rat brain, adjacent to, and sometimes overlapping sites of high density IGF-I receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-I and its autocrine role in growth of MCF-7 human breast cancer cells in culture.

Human MCF-7 breast cancer cells have been studied to determine their suitability as an autocrine model for the synthesis, secretion and action of insulin-like growth factor-I (IGF-I). Secretion of immunoreactive (ir-) IGF-I into serum-free medium was very low (less than 500 pg/10(6) cells per day). Northern blot hybridization detected at least two IGF-I messenger RNA transcripts (approximately 4.6 and approximately 1.8 kb) which were similar in size to those reported in other human and rat tissues. IGF-II mRNA was also detected but at low abundance. Cell proliferation was stimulated in a dose-responsive manner by exogenous IGF-I (10-30 ng/ml). Addition of a monoclonal antibody against IGF-I to MCF-7 cells in serum-free medium caused an inhibition of cell proliferation, suggesting that endogenous locally produced IGF-I does play an autocrine/paracrine role in MCF-7 cell growth. Proliferation of MCF-7 cells was sensitive to oestradiol (10 nM) in the absence but not in the presence of the weakly oestrogenic pH indicator phenol red. Neither IGF-I secretion nor IGF-I mRNA synthesis, however, was affected by addition of oestradiol. Similarly, GH, dexamethasone or dexamethasone plus oestradiol had no effect on either parameter. These data indicate that MCF-7 cells synthesize, secrete and respond to IGF-I. The very low levels of ir-IGF-I produced and their apparent lack of hormonal modulation suggest, however, that further studies are required to establish whether IGF-I plays a major physiological role in growth and development of MCF-7 cells.

Gestational- and labour-associated changes in the relative abundance of prostaglandin G/H synthase-1 and -2 mRNA in ovine placenta.

The aim of this study was to establish the gestational- and labour-associated variation in the relative abundance of prostaglandin synthase-1 (PGHS-1) and prostaglandin synthase-2 (PGHS-2) mRNA in ovine placenta (cotyledons). Cotyledons were collected from non-labouring ewes at 40-145 days of gestation (n = 25) and from ewes in active labour (145-147 days, n = 5). The relative abundance of PGHS-1 and PGHS-2 mRNA transcripts was determined by Northern blot analysis and laser densitometry, using a 2.3 kb sheep and a 1.2 kb mouse cDNA probe respectively. Data were expressed as a ratio of PGHS transcript hybridization/18S rRNA hybridization. During pregnancy, the relative abundance of PGHS-2 mRNA increased sevenfold, from 0.19 +/- 0.04 at 40-85 days (n = 5) to 1.39 +/- 0.05 at 140-145 days (n = 4) (P < 0.01). PGHS-1 mRNA relative abundance did not change significantly (P > 0.05) during gestation. Neither PGHS-1 nor PGHS-2 mRNA relative abundance changed significantly in association with labour onset at term (n = 5) when compared with the relative abundance observed at 140-145 days (n = 4) (P > 0.05). The data obtained in this study are consistent with the hypothesis that PGHS-1 is constitutively expressed in ovine placenta during pregnancy and at the time of labour, and that PGHS-2 is induced during the second half of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of the phospholipase A2 receptor messenger RNA in human gestational tissues.

Recently, a model has been proposed for the involvement of secretory phospholipase A2 (sPLA2) in the onset of and/or progression of human labour via the metabolism of cell membrane glycerophospholipids to generate biologically active, phospholipid-derived mediators. The recent molecular cloning and characterization of a cell-surface receptor for sPLA2 raise the possibility that sPLA2 enzymes may also affect cell function in intrauterine tissues via a receptor-mediated pathway. The aim of this study was to determine the expression profile of the PLA2 receptor messenger RNA in human gestational tissues at term. Messenger RNA for the PLA2 receptor was detected in amnion, choriodecidua and placenta by RT-PCR and transcripts of similar size to the 6.5- and 5.4-kb transcripts previously reported in various other human tissues were detected in choriodecidua by Northern blot analysis. However, smaller transcripts of approximately 4, 2.3 and 1 kb were also detected in choriodecidua by Northern blot analysis and the 2.3-kb transcript and the 1-kb transcript were the only major transcripts detected in amnion and placenta, respectively. The presence of PLA2 receptor mRNA in human gestational tissues indicates that an alternative non-catalytic pathway may contribute to the regulatory effects of sPLA2 isozymes in these tissues. While the specificity and affinity of the various transcripts identified in this study have yet to be determined, PLA2 isozymes released from human gestational tissues during pregnancy and at the time of labour may function as paracrine or autocrine mediators to affect cell function.

Staging of pancreatic cancer before and after neoadjuvant chemoradiation.

Neoadjuvant chemoradiation therapy is used at many institutions for treatment of localized adenocarcinoma of the pancreas. Accurate staging before neoadjuvant therapy identifies patients with distant metastatic disease, and restaging after neoadjuvant therapy selects patients for laparotomy and attempted resection. The aims of this study were to (1) determine the utility of staging laparoscopy in candidates for neoadjuvant therapy and (2) evaluate the accuracy of restaging CT following chemoradiation. Staging laparoscopy was performed in 98 patients with radiographically potentially resectable (no evidence of arterial abutment or venous occlusion) or locally advanced (arterial abutment or venous occlusion) adenocarcinoma of the pancreas. Unsuspected distant metastasis was identified in 8 (18%) of 45 patients with potentially resectable tumors and 13 (24%) of 55 patients with locally advanced tumors by CT. Neoadjuvant chemoradiation therapy and restaging CT were completed in a total of 103 patients. Thirty-three patients with potentially resectable tumors by restaging CT underwent surgical exploration and resections were performed in 27 (82%). Eleven (22%) of 49 patients with locally advanced tumors by restaging CT were resected, with negative margins in 55%; the tumors in these 11 patients had been considered locally advanced because of arterial involvement on restaging CT. Staging laparoscopy is useful for the exclusion of patients with unsuspected metastatic disease from aggressive neoadjuvant chemoradiation protocols. Following neoadjuvant chemoradiation, restaging CT guides the selection of patients for laparotomy but may overestimate unresectability to a greater extent than does prechemoradiation CT.

Predictive model for the diagnosis of intraabdominal abscess.

RATIONALE AND OBJECTIVES: The authors investigated the use of an artificial neural network (ANN) to aid in the diagnosis of intraabdominal abscess. MATERIALS AND METHODS: An ANN was constructed based on data from 140 patients who underwent abdominal and pelvic computed tomography (CT) between January and December 1995. Input nodes included data from clinical history, physical examination, laboratory investigation, and radiographic study. The ANN was trained and tested on data from all 140 cases by using a round-robin method and was compared with linear discriminate analysis. A receiver operating characteristic curve was generated to evaluate both predictive models. RESULTS: CT examinations in 50 cases were positive for abscess. This finding was confirmed by means of laboratory culture of aspirations from CT-guided percutaneous drainage in 38 patients, ultrasound-guided percutaneous drainage in five patients, surgery in five patients, and characteristic appearance on CT scans without aspiration in two patients. CT scans in 90 cases were negative for abscess. The sensitivity and specificity of the ANN in predicting the presence of intraabdominal abscess were 90% and 51%, respectively. Receiver operating characteristic analysis showed no statistically significant difference in performance between the two predictive models. CONCLUSION: The ANN is a useful tool for determining whether an intraabdominal abscess is present. It can be used to set priorities for CT examinations in order to expedite treatment in patients believed to be more likely to have an abscess.

Increased endoplasmic reticulum stress in decidual tissue from pregnancies complicated by fetal growth restriction with and without pre-eclampsia.

OBJECTIVES: Endoplasmic reticulum (ER) stress has been implicated in both pre-eclampsia (PE) and fetal growth restriction (FGR), and is characterised by activation of three signalling branches: 1) PERK-pEIF2α, 2) ATF6 and 3) splicing of XBP1(U) into XBP1(S). To evaluate the contribution of ER stress in the pathogenesis of PE relative to FGR, we compared levels of ER stress markers in decidual tissue from pregnancies complicated by PE and/or FGR. STUDY DESIGN: Whole-genome transcriptional profiling was performed on decidual tissue from women with PE (n = 13), FGR (n = 9), PE+FGR (n = 24) and controls (n = 58), and used for pathway and targeted transcriptional analyses of ER stress markers. The expression and cellular localisation of ER stress markers was assesses by Western blot and immunofluorescence analyses. RESULTS: Increased ER stress was observed in FGR and PE+FGR, including both the PERK-pEIF2α and ATF6 signalling branches, whereas ER stress was less evident in isolated PE. However, these cases demonstrated elevated levels of XBP1(U) protein. ATF6 and XBP1 immunoreactivity was detected in most (>80%) extravillous trophoblasts, decidual cells and macrophages. No difference in the proportion of immunopositive cells or staining pattern was observed between study groups. CONCLUSIONS: Increased PERK-pEIF2α and ATF6 signalling have been associated with decreased cellular proliferation and may contribute to the impaired placental growth characterising pregnancies with FGR and PE+FGR. XBP1(U) has been proposed as a negative regulator of ER stress, and increased levels in PE may reflect a protective mechanism against the detrimental effects of ER stress.

Objective prioritization of positional candidate genes at a quantitative trait locus for pre-eclampsia on 2q22.

Pre-eclampsia/eclampsia (PE/E) is a common, serious medical disorder of human pregnancy. Familial association of PE/E has been recognized for decades, but the genetics are complex and poorly understood. In an attempt to identify PE/E susceptibility genes, we embarked on a positional cloning strategy using 34 Australian and New Zealand PE/E pedigrees. An initial 10-cM resolution genome scan revealed a putative susceptibility locus spanning a broad region on chromosome 2 that overlaps an independently determined linkage signal seen in Icelandic PE pedigrees. Subsequent fine mapping using 25 additional short tandem repeat (STR) markers in this region and non-parametric multipoint linkage analysis did not change the overall position. Under a strict diagnosis of PE, we obtained significant evidence of linkage on 2q with a peak log-of-odds ratio score (LOD) of 3.43 near marker D2S151 at 155 cM. To prioritize positional candidate genes at the 2q locus for detailed analysis, we applied an objective prioritization strategy that integrates quantitative bioinformatics, assessment of differential gene expression and association analysis of single-nucleotide polymorphisms (SNPs). Highest priority was assigned to the activin receptor gene ACVR2. This gene also showed >10-fold differential gene expression in human decidual tissue from normotensive and PE individuals. We genotyped five known SNPs in this gene in our pedigrees and performed tests for association and linkage disequilibrium. One SNP (rs1424954) showed strong preliminary evidence of association with PE (P = 0.007), whereas two others (rs1364658 and rs1895694) exhibited nominal evidence (P < 0.05). Haplotype analysis revealed no additional association information. There was evidence of weak linkage disequilibrium among these SNPs. The highest observed LD occurred between the two strongest associated SNPs, suggesting that the observed signals may be the signature of an observed functional variant.

Detection of CAG repeats in pre-eclampsia/eclampsia using the repeat expansion detection method.

Pre-eclampsia/eclampsia is a serious disorder of human pregnancy that has a worldwide incidence of 2-10% and carries a severe morbidity and mortality risk for both mother and child. Its precise cause remains unknown. However, there is increasing evidence of an underlying complex maternal genetic susceptibility. Its high population incidence in the face of strong negative selection pressure suggests that the gene(s) involved have a selective advantage and/or a high mutation rate. One class of genetic diseases that involve a high mutation rate are the trinucleotide repeat expansion diseases. Thus, the aim of this study was to determine whether there is an association between a trinucleotide (CAG) repeat expansion and pre-eclampsia/eclampsia. We have used the repeat expansion detection (RED) method, which was developed to directly identify clinically significant repeat expansions, to analyse genomic DNA from an Australian and New Zealand population. The maximal CAG repeat length for each individual was recorded and the Mann-Whitney U and Wilcoxon rank sum test for independent samples were used to compare distributions for CAG/CTG repeats between two populations. There were no statistically significant differences between the distribution of CAG repeats in normotensive (n = 59) and severe pre-eclampsia (n = 69) (Mann-Whitney U = 1732; P = 0.14), and normotensive (n = 59) and eclamptic (n = 15) populations (Mann-Whitney U = 417, P = 0.726). Therefore, these RED results do not support a role for a large CAG expansion in pre-eclampsia/eclampsia. However, these data do not preclude the possibility that a small CAG expansion is associated with the disorder nor do they negate the hypothesis that a highly mutable gene contributes to the genetic component of pre-eclampsia/eclampsia.

Long-time dynamics of Met-enkephalin: comparison of theory with Brownian dynamics simulations.

A recent theory for the long time dynamics of flexible chain molecules is applied for the first time to a peptide of biological importance, the neurotransmitter met-enkephalin. The dynamics of met-enkephalin is considerably more complicated than that of the previously studied glycine oligomers; met-enkephalin contains the interesting motions of phenyl groups and of side chains relative to the backbone, motions that are present in general flexible peptides. The theory extends the generalized Rouse (GR) model used to study the dynamics of polymers by providing a systematic procedure for including the contributions from the memory function matrices neglected in the GR theory. The new method describes the dynamics by time correlation functions instead of individual trajectories. These correlation functions are analytically expressed in terms of a set of equilibrium averages and the eigenvalues and eigenfunctions of the diffusion operator. The predictions of the theory are compared with Brownian dynamics (BD) simulations, so that both theory and simulation use identical potential functions and solvent models. The theory thus contains no adjustable parameters. Inclusion of the memory function contributions profoundly affects the dynamics. The theory produces very good agreement with the BD simulations for the global motions of met-enkephalin. It also correctly predicts the long-time relaxation rate for local motions.

Identification and tissue distribution of messenger RNA for the growth hormone receptor in the rabbit.

Rabbit liver, a rich source of specific growth hormone (GH) receptors, contains three mRNA transcripts (4.2-4.5 kb, 3.1-3.2 kb, and 1.8-2.0 kb) which hybridize strongly to oligonucleotide probes complementary to nucleotide sequences in the extracellular and cytoplasmic regions of the rabbit liver GH receptor. The approximately 4.5 kb transcript was the most abundant and showed some sex difference (male greater than female) and a significant, approximately 2 fold increase in late pregnancy - observations consistent with changes seen in the specific 125I-hGH binding capacity of rabbit liver membranes prepared from the same tissue samples. The approximately 4.5 kb mRNA species, but not the smaller transcripts, was also detected, at lower abundance, in rabbit kidney, heart and lung but not in mammary gland, which is known to lack 125I-GH binding activity. These studies have identified the nature of the mRNA transcripts coding for the GH receptor in recognized/potential GH target tissues in the rabbit. The regulation of the major GH receptor mRNA in rabbit liver appears to broadly reflect known changes in expressed receptor protein.

MRI of conjoined twins illustrating advances in fetal imaging.

Complex congenital fetal abnormalities are optimally evaluated with both ultrasound and magnetic resonance imaging. With the advent of ultrafast imaging sequences, fetal imaging has significantly improved. We present two cases of conjoined thoracoomphalopagus twins to discuss this unusual anomaly and to illustrate the recent advances in magnetic resonance imaging of the fetus.

Isolated pelvic deep venous thrombosis: relative frequency as detected with MR imaging.

PURPOSE: To determine the relative frequency of deep venous thrombosis (DVT) isolated to the pelvic veins, as demonstrated with magnetic resonance (MR) imaging. MATERIALS AND METHODS: The reports of 769 MR examinations performed from June 1993 through December 1999 in patients with suspected DVT were reviewed retrospectively. MR venography was performed by using a two-dimensional gradient-recalled-echo sequence (typically repetition time, 34 msec; echo time, 13 msec; flip angle, 60 degrees ). The presence of DVT was categorized by location in the pelvis, thigh, or calf. RESULTS: DVT was identified in 167 (21.7%) of the 769 MR examinations. Thirty-four (20.4%) of the 167 studies demonstrated DVT isolated to the pelvic veins. CONCLUSION: The relative frequency of isolated pelvic DVT detected with MR venography was higher than that reported in prior studies with ultrasonography (US) or ascending venography. MR venography should be performed in patients with suspected pelvic DVT or when clinical suspicion persists despite a negative US study.

Vaginal opacification during defecography: direction of vaginal migration aids in diagnosis of pelvic floor pathology.

BACKGROUND: To determine whether direction of vaginal displacement during defecography aids in diagnosing pelvic floor pathology. METHODS: Ninety patients underwent defecography over a 2-year period. Each study was retrospectively reviewed by three radiologists who recorded whether the vagina was displaced cephalad, caudad, or nondisplaced in relation to the urogenital hiatus. This information was then correlated with radiologic diagnosis rendered for the study. RESULTS: Of the 26 patients with normal defecograms, 19 (73%; p < 0. 001) demonstrated no vaginal displacement during the procedure. Comparatively, 10 (83%; p < 0.001) of the 12 patients with cystoceles showed caudad vaginal displacement, and no patients with cystoceles showed cephalad displacement of the vagina. Of the 17 patients with rectoceles, 10 (58%) showed cephalad displacement, one (6%) showed caudad displacement, and six (35%) patients showed no vaginal displacement. Thirteen (46%) of 28 patients with enteroceles showed cephalad vaginal displacement, nine (32%) showed no vaginal displacement, and six (21%) demonstrated caudad displacement. CONCLUSIONS: Caudad displacement of the opacified vagina suggests the presence of a cystocele. Cephalad vaginal displacement is suggestive of the presence of an enterocele or rectocele.

Vaginal opacification during defecography: utility of placing a folded gauze square at the introitus.

We evaluated the value of placement of a folded gauze square into the urogenital introitus to improve vaginal opacification in 90 patients who underwent defecography. Of the 50 patients who retained the gauze in the introitus, 96% demonstrated excellent or good vaginal opacification. By contrast, only 75% of the 40 patients who lost the gauze during the study were able to achieve the same level of opacification. This difference was shown to be statistically significant (p < 0.002), suggesting that placement of a folded gauze square in the introitus limits loss of contrast from the vagina, which improves vaginal opacification.

An enzyme-linked immunosorbent assay (ELISA) for in vitro pollen growth based on binding of a monoclonal antibody to the pollen tube surface.

An indirect method of quantifying in vitro pollen growth has been developed. This is based on the finding that a monoclonal antibody (PCBC3), which has a primary specificity for alpha-l-arabinofuranosyl residues, binds to the surface of in vitro grown pollen tubes of the ornamental tobacco, Nicotiana alata. This binding was quantified using an enzyme linked immunosorbent assay (ELISA). The method was used to determine the effects of 2-deoxy-d-glucose and nonanoic acid on in vitro pollen growth.