Construction of a tissue microarray with two millimeters cores of endometrioid endometrial cancer: factors affecting the quality of the recipient block

The tissue microarray (TMA) method currently is not used to render a primary diagnosis of cancer, but its scientific value has been proved in studies of various cancer types. TMA technology still is not used often for uterine tumors, however. We investigated the repeatability of histological diagnosis of endometrioid endometrial cancer (EEC) using conventional histology and TMA using 2 mm cores. We examined EEC tissues from 171 patients. Formalin fixed, paraffin embedded tissue donor blocks from EEC specimens were selected and examined histologically. Duplicate 2 mm tissue cores were inserted into a TMA recipient block. EEC tissues were examined as hematoxylin-eosin stained sections from the TMAs. EEC tissue was identified in the TMAs in 158 cases (92.4%) and not found in 13 cases (7.6%). On the TMA slides, both EEC positive cores were identified in 129 cases (75.4%), but only one core in 29 cases (17.0%). Among 342 biopsies of the donor blocks (each case in duplicate), EEC was found in 287 cases (83.9%) using the TMA: 124/146 (84.9%) with superficial infiltration, 153/178 (86.0%) with deep myometrial infiltration, and 10/18 (55.6%) without myometrial infiltration. We concluded that two 2 mm tissue cores from a biopsy of a donor block inserted into a TMA recipient block were sufficient to diagnose EEC in more than 90% of cases. EEC was identified in the TMAs with similar frequency with respect to superficial and deep myometrial infiltration. Cases without myometrial infiltration were identified less often.     

A theory for the chemical mediation of the excitability of the brain, with special reference to natural and drug-induced sleep.

We review evidence which suggests to us that uracil, or a molecule structurally resembling uracil, is likely to be involved in the mediation of level of arousal. In addition we attempt to show that at least one theory, based on this idea, provides a molecular mechanism for certain features of natural sleep, and for the effect on arousal of several classes of drugs and of certain gene mutations. This theory, and related ones, can feasibly be tested further with existing methods.     

A general approach to proving the minimality of phylogenetic trees illustrated by an example with a set of 23 vertebrates

Summary We have recently described a method of building phylogenetic trees and have outlined an approach for proving whether a particular tree is optimal for the data used. In this paper we describe in detail the method of establishing lower bounds on the length of a minimal tree by partitioning the data set into subsets. All characters that could be involved in duplications in the data are paired with all other such characters. A matching algorithm is then used to obtain the pairing of characters that reveals the most duplications in the data. This matching may still not account for all nucleotide substitutions on the tree. The structure of the tree is then used to help select subsets of three or more. characters until the lower bound found by partitioning is equal to the length of the tree. The tree must then be a minimal tree since no tree can exist with a length less than that of the lower bound.The method is demonstrated using a set of 23 vertebrate cytochrome c sequences with the criterion of minimizing the total number of nucleotide substitutions. There are 131130 7045768798 9603440625 topologically distinct trees that can be constructed from this data set. The method described in this paper does identify 144 minimal tree variants. The method is general in the sense that it can be used for other data and other criteria of length. It need not however always be possible to prove a tree minimal but the method will give an upper and lower bound on the length of minimal trees.     

A scanning electron-microscopic study of the rat thymus with special reference to cell types and migration of lymphocytes into the general circulation

Summary The three-dimensional structure of the rat thymus was studied by combined scanning and transmission electron microscopy. The thymus consists mainly of four types of cells: epithelial cells, lymphocytes, macrophages, and interdigitating cells (IDCs).The epithelial cells form a meshwork in the thymus parenchyma. Cortical epithelial cells are stellate in shape, while the medullary cells comprise two types: stellate and large vacuolated elements. A continuous single layer of epithelial cells separates the parenchyma from connective tissue formations of the capsule, septa and vessels. Surrounding the blood vessels, this epithelial sheath is continuous in the cortex, while it is partly interrupted in the medulla, suggesting that the blood-thymus barrier might function more completely in the cortex.Cortical lymphocytes are round and vary in size, whereas medullary lymphocytes are mainly small, although they vary considerably in surface morphology.Two types of large wandering cells, macrophages and IDCs, could be distinguished, as well as intermediate forms. IDCs sometimes embraced or contacted lymphocytes, suggesting their role in the differentiation of the latter cells.Perivascular channels were present around venules and some arterioles in the cortico-medullary region and in the medulla. A few lymphatic vessels were present in extended perivascular spaces.The present study suggests the possible existence of two routes of passage of lymphocytes into the general circulation. One is via the lymphatics, while the other is through the postcapillary venules into the blood circulation. Our SEM images give evidence that lymphocytes use an intracellular route, i.e., the endothelium of venules.     

A polymerase chain reaction-based approach to cloning sigma factors from eubacteria and its application to the isolation of a sigma-70 homolog from Chlamydia trachomatis.

Taking advantage of the known sequence conservation of portions of bacterial sigma factor proteins, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction (PCR) to amplify DNA sequences from the chlamydial genome. The PCR products were used as a probe to recover the genomic fragments from a library of cloned murine Chlamydia trachomatis DNA. Sequence analysis of one of these clones revealed striking homology to the sigma-70 protein of Escherichia coli and the sigma-43 protein of Bacillus subtilis, strongly implying that this locus (sigA) encodes the major vegetative sigma factor of murine C. trachomatis. This PCR-based approach will be broadly applicable to the cloning of major sigma factors from other eubacteria.     

A new opuntioid cactus from the Cayman Islands, B.W.I., with a discussion and key to the genus Consolea Lemaire

A new subspecies ofConsolea millspaughii is described from the eastern bluff of Cayman Brac and northern coastal terrace of Little Cayman (Cayman Islands, British West Indies). This taxon has been mistakenly identified asConsolea spinosissima since its first collection in 1938. However, the smooth surface of its stem segments, not being demarcated by a network of depressed lines, its pitted areoles, and the fairly reduced pedicellate area of the pericarpel set is apart from this Jamaican endemic. Because the novelty here described brings to three the number ofConsolea taxa that are characterized by very closely set, pitted areoles, an informal “C. millspaughii species-group” is recognized. This assemblage of Western Caribbean opuntioids includesConsolea corallicola, C. millspaughii subsp.millspaughii, andC. millspaughii subsp.caymannnsis. The new subspecies is described, illustrated, and compared withC. spinosissima and with kindred taxa within the species-group. A key to all species and subspecies ofConsolea is provided.     

A new general method for the assessment of the molecular-weight distribution of polydisperse preparations. Its application to an intestinal epithelial glycoprotein and two dextran samples, and comparison with a monodisperse glycoprotein

A specimen of intestinal glycoprotein isolated from the pig and two samples of dextran, all of which are polydisperse (that is, the preparations may be regarded as consisting of a continuous distribution of molecular weights), have been examined in the ultracentrifuge under meniscus-depletion conditions at equilibrium. They are compared with each other and with a glycoprotein from Cysticercus tenuicollis cyst fluid which is almost monodisperse. The quantity c(-(1/3)) (c=concentration) is plotted against xi (the reduced radius); this plot is linear when the molecular-weight distribution approximates to the ;most probable', i.e. when M(n):M(w):M(z): M((z+1))....... is as 1:2:3:4: etc. The use of this plot, and related procedures, to evaluate qualitatively and semi-quantitatively molecular-weight distribution functions where they can be realistically approximated to Schulz distributions is discussed. The theoretical basis is given in an Appendix.     

A new approach to antiglaucoma drugs: carbonic anhydrase inhibitors with or without NO donating moieties. Mechanism of action and preliminary pharmacology

The clinically used sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor dorzolamide (DRZ), a new sulfonamide CA inhibitor also incorporating NO-donating moieties, NCX250, and isosorbide mononitrate (ISMN) (an NO-donating compound with no CA inhibitory properties) were investigated for their intraocular pressure (IOP) lowering effects in rabbits with carbomer-induced glaucoma. NCX250 was more effective than DRZ or ISMN on lowering IOP, increasing ocular hemodynamics, decreasing the inflammatory processes and ocular apoptosis in this animal model of glaucoma. NO participate to the regulation of IOP in glaucoma, having also antiapoptotic and anti-inflammatory effects. The ophthalmic artery, both systolic and diastolic velocities, were significantly reduced in NCX250-treated eyes in comparison to DRZ treated ones, suggesting thus a beneficial effect of NCX250 on the blood supply to the optic nerve. Combining CA inhibition with NO-donating moieties in the same compound offers an excellent approach for the management of glaucoma.     

A new experimental approach to detect long-range conformational changes transmitted between the membrane and cytosolic domains of LmrA,a bacterial multidrug transporter

LmrA confers multidrug resistance to Lactococcus lactis by mediating the extrusion of antibiotics, out of the bacterial membrane, using the energy derived from ATP hydrolysis. Cooperation between the cytosolic and membrane-embedded domains plays a crucial role in regulating the transport ATPase cycle of this protein. In order to demonstrate the existence of a structural coupling required for the cross-talk between drug transport and ATP hydrolysis, we studied specifically the dynamic changes occurring in the membrane-embedded and cytosolic domains of LmrA by combining infrared linear dichroic spectrum measurements in the course of H/D exchange with Trp fluorescence quenching by a water-soluble attenuator. This new experimental approach, which is of general interest in the study of membrane proteins, detects long-range conformational changes, transmitted between the membrane-embedded and cytosolic regions of LmrA. On the one hand, nucleotide binding and hydrolysis in the cytosolic nucleotide binding domain cause a repacking of the transmembrane helices. On the other hand, drug binding to the transmembrane helices affects both the structure of the cytosolic regions and the ATPase activity of the nucleotide binding domain.     

Purification of a growth factor related to platelet-derived growth factor and a type beta transforming growth factor secreted by mouse neuroblastoma cells. A general strategy for the purification of basic polypeptide growth factors.

A general strategy was developed for the purification of basic polypeptide growth factors. This method is a combination of gel filtration, weak-cation-exchange h.p.l.c. and reverse-phase h.p.l.c., separating the proteins according to size, charge and hydrophobicity respectively. All steps are carried out at low pH with exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by freeze-drying facilitate the detection of the growth factors by biological assays. By using this method, homogeneous preparations of two basic growth factors were purified in high yield from mouse-neuroblastoma-Neuro-2A-cell-conditioned medium. It is shown that these purified factors are biochemically and immunologically related to platelet-derived growth factor and type beta transforming growth factor from human platelets.     

Fine structure of two Hygropetra species,Hygropetra gelasina sp. nov. and Hygropetra balfouriana (Bacillariophyceae), and the taxonomic position of the genus with special reference to Frankophila

A small diatom, Hygropetra gelasina sp. nov., obtained from wet moss is described. This diatom is similar to Hygropetra balfouriana (Grunow ex Cleve) Krammer & Lange‐Bertalot, which was found in the same moss sample. Fine structural observations revealed that H. gelasina has a reduced raphe slit and depressions along the margin of the axial area, at the proximal ends of the striae. Both species are characterized by multiple rows of areolae in each stria and a hexagonal pattern of sub‐pores similar to that in Pinnularia, but differing in the position of the areola occlusions or hymenes, which are internal in Hygropetra. Comparison with Frankophila, which shares the characteristics of reduced raphe slits and areola structure with Hygropetra, provides a reference for future taxonomic study of these related genera.     

External characteristics and associated developmental changes in two species of Sulawesi macaques,Macaca tonkeana andM. hecki,with special reference to hybrids and the borderland between the species

The degree of intergradation between two species of Sulawesi macaques,Macaca tonkeana andM. hecki, was studied by examining the diagnostic external characteristics of more than 100 monkeys kept as pets by natives. Two possible hybrid monkeys were found and both originated from the borderland between the two species, located in the most proximal region of the northern peninsula of Sulawesi. The previously postulated wide area of integradation between the two species at the possible contact zone was, however, not recognized, and typical examples oftonkeana orhecki were found to be present on the two sides of a narrow “hybrid” zone which was defined by direct observations. Furthermore, despite considerable individual variations, we were able to allocate most monkeys to one or other of the species. Each of ten external characteristics of the members of both species more or less encompassed the individual variations, but may undergo changes with the development of the monkeys. The mechanisms of reproduction of hybrid monkeys and the maintenance of differences between the species are discussed.     

A study on the renaturation of membrane proteins after solubilization in SDS or following polyacrylamide gel electrophoresis in SDS,with special reference to a phosphatase from acholeplasma laidlawii

It may be easier to renature SDS-denatured hydrophobic proteins than to renature SDS-denatured water-soluble proteins. This paper presents some support for this hypothesis in the form of literature reports and an experiment of our own with an intrinsic membrane protein (a phosphatase from Acholeplasma laidlawii), that could be completely renatured, to judge from the restored activity, which was equal to (or higher than) that of the untreated enzyme. If this hypothesis is correct it might be possible to devise general methods to reverse the SDS denaturation of hydrophobic membrane proteins. This would be a breakthrough in the purification of at least some membrane proteins, because the high-resolving polyacrylamide gel electrophoresis in SDS could then be used to prepare membrane proteins in a native state. The method used for the renaturation of the SDS-denatured, entirely inactive, phosphatase comprised removal of SDS with the aid of conventional dialysis against a buffer containing the neutral, very efficient and non ultraviolet light-absorbing detergent G3707. For renaturation of the enzyme following an SDS-electrophoresis in polyacrylamide the gel was immersed in the same buffer for several hours; by staining for phosphatase the enzyme could easily be localized in the gel in the form of a yellow band, coinciding with a protein zone.     

New general approach for determining the solution structure of a ligand bound weakly to a receptor: structure of a fibrinogen Aalpha-like peptide bound to thrombin (S195A) obtained using NOE distance constraints and an ECEPP/3 flexible docking program.

A new approach incorporating flexible docking simulations and NMR data is presented for calculating the bound conformation of a ligand that interacts weakly with an enzyme. This approach consists of sampling directly the conformation of a flexible ligand inside a receptor active site containing surrounding flexible loops. To make this sampling efficient, a ligand-growing procedure has been adopted. Optimization of the ECEPP/3-plus-NOE constraint function is carried out by using a collective variable Monte Carlo minimization technique. Numerous energy minimizations are made possible for such a large system by using a Bezier splines energy grid technique. This new flexible docking approach was applied to determine the structure of a fibrinogen Aalpha-like peptide (7DFLAEGGGVRGPRV20) bound to an active site mutant of thrombin [thrombin(S195A)]. Structure calculations of the bound ligand, using 2D-transferred NOESY distance constraints in the DIANA program, showed that the N-terminal portion of the peptide (D7-R16) involves a chain reversal, whereas the C-terminal portion (G17-V20) adopts a fold that exists in several different orientations. In addition, the ECEPP/3 flexible docking package was used to assess the conformational variability of the ligand and surrounding 60D-insertion loop of thrombin. Amino acid residues (17-20) of the peptide interact with a region of the enzyme that exhibits broad specificity, with a preferred direction between the 60D-insertion loop and Pro37 of thrombin.     

Compound design guidelines for evading the efflux and permeation barriers of Escherichia coli with the oxazolidinone class of antibacterials: Test case for a general approach to improving whole cell Gram-negative activity

Previously we reported the results from an effort to improve Gram-negative antibacterial activity in the oxazolidinone class of antibiotics via a systematic medicinal chemistry campaign focused entirely on C-ring modifications. In that series we set about testing if the efflux and permeation barriers intrinsic to the outer membrane of Escherichia coli could be rationally overcome by designing analogs to reside in specific property limits associated with Gram-negative activity: i) low MW (<400), ii) high polarity (clogD_7.4 <1), and iii) zwitterionic character at pH 7.4. Indeed, we observed that only analogs residing within these limits were able to overcome these barriers. Herein we report the results from a parallel effort where we explored structural changes throughout all three rings in the scaffold for the same purpose. Compounds were tested against a diagnostic MIC panel of Escherichia coli and Staphylococcus aureus strains to determine the impact of combining structural modifications in overcoming the OM barriers and in bridging the potency gap between the species. The results demonstrated that distributing the charge-carrying moieties across two rings was also beneficial for avoidance of the outer membrane barriers. Importantly, analysis of the structure-permeation relationship (SPR) obtained from this and the prior study indicated that in addition to MW, polarity, and zwitterionic character, having ≤4 rotatable bonds is also associated with evasion of the OM barriers. These combined results provide the medicinal chemist with a framework and strategy for overcoming the OM barriers in GNB in antibacterial drug discovery efforts.     

The right to informed choice. A study and opinion poll of women who were or were not given the option of a sterilisation with their caesarean section

Background

In the Netherlands, caesarean sections (CSs) are rarely combined with tubal occlusion (TO), partly because discussing CS/TO near delivery is considered unethical and earlier hypothetical counselling – i.e. suppose you happen to need a CS – is rare. This results in more unintended pregnancies and is inconsistent with informed choice. We explored whether TO should indeed not be made routinely available to eligible women.

Methods and Findings

A questionnaire was mailed to 515 Para ≥2 who underwent in the past ≥1 CS. 498 (96.7%) responded. They were on average 35.3 years old, had 2.5 children, had undergone 1.6 CSs, and 3.3 years had passed since their index delivery, either a CS (393) or vaginal birth (105) after a previous CS. 87% of the 498 believed that pregnant mothers with ≥1 children should be routinely counselled about CS/TO. Indeed, 58% and 85% respectively, thought women/couples expecting their second or third child should still be given the TO option days before delivery, if omitted earlier. Counselled women, 138/498 (27.8%), were far more often satisfied than those without CS/TO option. 33/393 had a CS/TO. None indicated regret in the questionnaire. Another 119 also would have elected a CS/TO if given that option. Therefore, 152 (38.7%) of 393 Para ≥2 had or would have liked a concurrent TO. 118/119 wrote they still regretted missing this opportunity. The exception''s husband had had a vasectomy. 100/119 were good TO candidates: they were ≥28 years when they delivered an apparently healthy baby of ≥37 weeks. The current contraceptive use of these 100 suggests that this group will have at least 8 unintended pregnancies before age 50.

Conclusion

The experiences and opinions of previous potential candidates for a CS/TO do not support the reluctance of Dutch obstetricians to counsel pregnant Para ≥1 about the TO option for a (potential) CS.     

Heteroclitic polyclonal and monoclonal anti-Gm(a) and anti-Gm(g) human rheumatoid factors react with epitopes induced in Gm(a-), Gm(g-) IgG by interaction with antigen or by nonspecific aggregation. A possible mechanism for the in vivo generation of rheumatoid factors.

Heteroclitic rheumatoid factors (RF) are specific for allotypic determinants, e.g., Gm(a) or Gm(g) on allogeneic, but not autologous IgG. All polyclonal RF we isolated from nine rheumatoid arthritis patients with circulating Gm(a-), (b+), (g-), (f+) IgG displayed dual heteroclitic activity for the Gm(a) and Gm(g) allotypes, as shown by using appropriate RBC agglutination assays and affinity columns bearing Gm(a+) or Gm(g+) IgG. To investigate possible mechanisms underlying the in vivo generation of heteroclitic RF, we tested the ability of nonspecifically and immune-specifically aggregated Gm(a-), (g-) IgG to function as targets for RF from Gm(a-), (g-) patients with rheumatoid arthritis. Heat aggregation (63 degrees C for 20 min) or binding to Ag (as in tetanus toxoid-antitetanus toxoid complexes) induced a "functional" Gm(a+) and/or (g+) phenotype in Gm(a-), (g-) IgG from five healthy subjects and five rheumatoid patients, as suggested by the ability of these altered IgG to function as efficient targets for six heteroclitic RF in direct binding and competitive inhibition experiments. That heterocliticity and dual Gm(a), Gm(g) specificity can be features of a single antibody molecule was formally demonstrated by analysis of a monoclonal RF (IgM mAb 61) generated from a Gm(a-), (g-) rheumatoid patient. RF mAb 61 displayed a high affinity (Kd, 10(-7) M) for IgG Fc fragment of Gm(a+) and (g+) IgG or aggregated autologous Gm(a-), (g-) IgG but did not bind to native autologous IgG. To investigate the molecular basis of the acquired Gm(a) phenotype, PBMC from five Gm(a-) patients with rheumatoid arthritis and two Gm(a-) normal subjects arthritis and two Gm(a-) normal subjects were cultured in vitro after activation with PWM. In most instances, these PBMC produced IgG that behaved as Gm(a+) in sensitive ELISA. Application of the polymerase chain reaction (PCR), using probes specific for the nucleotide sequence coding for the Gm(a) tetrapeptide, to the amplification of DNA from the in vitro-stimulated Gm(a-) normals or rheumatoid patients' PBMC provided no evidence for Gm(a) nucleotide sequences. The present data suggest that acquisition of the Gm(a) determinant by Gm(a-) IgG may result from subtle changes in the CH2-CH3 RF-binding region. Such changes would occur when Gm(a) IgG are complexed with Ag or nonspecifically altered, thereby providing a possible explanation for the induction of heteroclitic RF in Gm(a-) rheumatoid arthritis patients.