Optimizing form and function with the direct posterior composite resin: a case report

The human testis determining factor (SRY) has been cloned from the Y chromosome. This gene is a dominant inducer of male differentiation. Mutations in the SRY gene result in an XY individual developing as a sex reversed phenotypic female. Sex reversal in humans can also be caused by mutations located in autosomal or X-linked loci. One such sex-reversing locus (SRAI) is associated with the developmental disorder campomelic dysplasia (CD). Both these syndromes were mapped to human chromosome 17q by the identification of balanced reciprocal translocations in five unrelated patients. The translocation breakpoint of one such XY-female CD patient was mapped and the region surrounding it cloned. The closest distal marker used to map the translocation breakpoint was the SOX9 gene. Because of the close proximity of this gene to the breakpoint, it was subjected to mutation analysis in patients without overt chromosome rearrangements. Analysis of DNA from these patients and their parents identified de novo mutations in the SOX9 gene in patients with both autosomal sex reversal and CD. This showed that mutations in the SOX9 gene are responsible for both syndromes.     

Food contamination from epoxy resins and organosols used as can coatings: analysis by gradient NPLC

Normal phase LC with gradient elution enabled the analysis of a broadened range of oligomers of BADGE (Bisphenol-A diglycidyl ether) and Novolak compounds in canned foods, such as sea foods in oil, meat products and soups. A major component released from Bisphenol-A resins was identified as the cyclo-(Bisphenol-A monoglycidyl ether) dimer and was commonly present in foods at concentrations of around 1 mg/kg. For the epoxy Novolaks, concentrations of the three- to six-ring compounds often far exceeded those of BFDGE (Bisphenol-F diglycidyl ether) and reached 20 mg/kg in foods. A two-step acylation is proposed for the detection of epoxy components.     

Molecular mass determination by sedimentation velocity experiments and direct fitting of the concentration profiles

A large English pedigree in which heterocellular hereditary persistence of fetal hemoglobin (HPFH) segregates is described. beta-globin cluster deletions and gamma gene promoter mutations associated with HPFH have been excluded. Of particular importance in this pedigree is the absence of any cosegregating hemoglobinopathy, thus allowing observation of the segregation pattern of this form of HPFH without the complicating effect of a beta-globin gene mutation. Information gained in this study confirms that the extent of elevation of hemoglobin (Hb) F and F cells varies between affected individuals. There are one example of incomplete penetrance and three examples of father-to-son transmission, thus excluding X-linked inheritance. Consistent with previous reports, the most likely mode of inheritance is autosomal codominant. Linkage studies using a beta-globin cluster microsatellite show no evidence of linkage to this chromosomal region implicating the presence of trans-acting regulatory factor(s). We have recently mapped one such locus to the chromosome 6q region in a very large Asian-Indian pedigree. Linkage to chromosome 6q in the English pedigree was excluded, thus indicating the presence of genetic heterogeneity in heterocellular HPFH.     

Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase

The ability to generate specific genetic modifications in mice provides a powerful approach to assess gene function. When genetic modifications have been generated in the germ line, however, the resulting phenotype often only reflects the first time a gene has an influence on - or is necessary for - a particular biological process. Therefore, systems allowing conditional genetic modification have been developed (for a review, see [1]); for example, inducible forms of the Cre recombinase from P1 phage have been generated that can catalyse intramolecular recombination between target recognition sequences (loxP sites) in response to ligand [2] [3] [4] [5]. Here, we assessed whether a tamoxifen-inducible form of Cre recombinase (Cre-ERTM) could be used to modify gene activity in the mouse embryo in utero. Using the enhancer of the Wnt1 gene to restrict the expression of Cre-ERTM to the embryonic neural tube, we found that a single injection of tamoxifen into pregnant mice induced Cre-mediated recombination within the embryonic central nervous system, thereby activating expression of a reporter gene. Induction was ligand dependent, rapid and efficient. The results demonstrate that tamoxifen-inducible recombination can be used to effectively modify gene function in the mouse embryo.     

Sensing by intrahepatic muscarinic nerves of a portal-arterial glucose concentration gradient as a signal for insulin-dependent glucose uptake in the perfused rat liver

In vivo, insulin increases net hepatic glucose uptake efficiently only in the presence of a portal-arterial glucose gradient. In isolated perfused rat livers supplied with a glucose gradient (portal 10 mM/arterial 5 mM) insulin-induced glucose uptake was blocked by atropine; in livers not supplied with the gradient (portal = arterial 5 mM) insulin-dependent glucose uptake was elicited by acetylcholine. Apparently, the gradient was sensed and transformed into a metabolic signal by intrahepatic nerves, releasing acetylcholine to muscarinic receptors.     

Lack of coactivator interaction can be a mechanism for dominant negative activity by mutant thyroid hormone receptors

We studied the interactions of two natural thyroid hormone receptor (TR) mutants from patients with resistance to thyroid hormone (RTH) and an artificial TR mutant with a nuclear receptor corepressor, N-CoR, and a steroid receptor coactivator, SRC-1. In electrophoretic mobility shift assays, wild-type TRbeta-1 interacted with N-CoR in the absence of ligand, whereas T3 caused dissociation of the TRbeta-1/N-CoR complex and formation of TRbeta-1/SRC-1 complex. In contrast, a natural mutant (G345R) with poor T3-binding affinity formed TRbeta-1/N-CoR complex, both in the absence and presence of T3, but could not form TRbeta-1/SRC-1 complex. Another TR mutant, which bound T3 with normal affinity and containing a mutation in the AF-2 region (E457D), had normal interactions with N-CoR but could not bind SRC-1. Both these mutants had strong dominant negative activity on wild-type TR transactivation. Studies with a TR mutant that had slightly decreased T3-binding affinity (R320H) showed a T3-dependent decrease in binding to N-CoR and increase in binding to SRC-1 that reflected its decreased ligand binding affinity. Additionally, when N-CoR and SRC-1 were added to these receptors at various T3 concentrations in electrophoretic mobility shift assays, TR/N-CoR and TR/SRC-1 complexes, but not intermediate complexes were observed, suggesting that N-CoR release is necessary before SRC-1 binding to TR. Our data provide new insight on the molecular mechanisms of dominant negative activity in RTH and suggest that the inability of mutant TRs to interact with coactivators such as SRC-1, which results from reduced T3-binding affinity, is a determinant of dominant negative activity.     

Numerical calculation of concentration distribution and dimensional change for constant-rate diffusion of a solute into a Solvent’s surface

Modified coordinate systems, such as have been utilized in the determination of mutual diffusion coefficients where volume changes occur, have been utilized in solving for concentration distributions and dimensional changes resulting from diffusion of a solute into a substrate solvent’s surface. Developed equations have been solved, utilizing simple explicit finite difference methods, for two example cases. In both examples the diffusion coefficient was assumed a function of concentration, surface flux was assumed constant, and temperature was assumed constant. Aluminum was assumed to be diffused into a copper substrate. The ξ _Y coordinate, containing an equal number of mols of the substrate component per increment, was found to be convenient for the case of a flat plate or cylindrical substrate. Results have been presented graphically. This treatment is applicable to single-phase systems wherein the arrival rate of solute atoms at the substrate surface, either by ionic transport in high-temperature electrodiffusion cells or from the vapor phase, is equal to the diffusion rate into the surface.     

Cell density-sensing in Dictyostelium by means of the accumulation rate, diffusion coefficient and activity threshold of a protein secreted by starved cells

The simple eukaryote Dictyostelium discoideum grows as an amoeba on leaf and soil surfaces. When starved, the amoebae aggregate and differentiate. The amoebae can also be induced to differentiate as isolated cells submerged in buffer, if the buffer contains a sufficiently high concentration of a protein (CMF) secreted by starved cells. CMF is also necessary for aggregation and differentiation on surfaces. This indicated that CMF has either an autocrine function or is part of a density-sensing system. To distinguish between these two possible functions, we first examined the rate at which CMF is accumulated and the activity threshold of cells for CMF, since both parameters will affect whether a cell can provide enough CMF to self-stimulate. We find that CMF potentiates its own accumulation, and that otherwise the accumulation rate and activity threshold are affected very little by a variety of physiological conditions. We then use diffusion calculations to show that even after many hours of continuous secretion, the CMF concentration adjacent to an isolated starved cell on a leaf or soil surface will be too low to allow differentiation, whereas an extracellular concentration of CMF sufficiently high to allow differentiation will occur when starved cells are at high densities. We find a close match between the predicted and experimentally observed density necessary for differentiation. The theoretical and observed behavior of cells at different cell densities suggests that due to its accumulation rate, diffusion coefficient, and activation threshold, CMF can function as part of a cell density-sensing system which allows Dictyostelium cells in the wild to co-ordinate their development.     

Place navigation in rats guided by a vestibular and kinesthetic orienting gradient.

The role of the slope of terrain in orientation was examined in rats trained to find, among 4 equidistant feeders, the 1 located in the upper left quadrant of a 10% tilted arena (1-m radius). Rats started from the center in light and with randomly changing slope direction reached the correct goal in 90% of 1st choices after 29 sessions. The same rats maintained 83% correct choices when the experiment was conducted in darkness. On a horizontal arena, their performance became random. After training, successful navigation was also observed (71% correct 1st choices) when the rats were started from different points at about 30 cm from the wall. This finding suggests that the slope of terrain may be used to establish a cognitive map based primarily on kinesthetic and vestibular signals. The flexibility of such a map seems to be rather limited, however, because changing the goal position with respect to inclination requires prolonged retraining. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Potent CD14-mediated signalling of human leukocytes by Escherichia coli can be mediated by interaction of whole bacteria and host cells without extensive prior release of endotoxin

How invading microorganisms are detected by the host has not been well defined. We have compared the abilities of Escherichia coli and lipopolysaccharides (LPS) purified from these bacteria to prime isolated neutrophils for phorbol myristate acetate-stimulated arachidonate release, to trigger respiratory burst in 1% blood, and to increase steady-state levels of tumor necrosis factor alpha mRNA in whole blood. In all three assays, bacteria were > or = 10-fold more potent than equivalent amounts of LPS and could trigger maximal cellular responses at ratios as low as one bacterium per 20 to 200 leukocytes. Both E. coli and LPS-triggered responses were enhanced by LPS-binding protein and inhibited by an anti-CD14 monoclonal antibody and the bactericidal/permeability-increasing protein (BPI). However, whereas O polysaccharide did not affect the potency of isolated LPS, intact E. coli carrying long-chain LPS (O111:B4) was less potent than rough E. coli (J5). Furthermore, material collected by filtration or centrifugation of bacteria incubated under conditions used to trigger arachidonate release or chemiluminescence was 5- or 30-fold less active, respectively, than whole bacterial suspensions. Extracellular BPI (not bound to bacteria) inhibited bacterial signalling, but BPI bound to bacteria was much more potent. Taken together, these findings indicate that E. coli cells can strongly signal their presence to human leukocytes not only by shedding LPS into surrounding fluids but also by exposing endotoxin at or near their surface during direct interaction with host cells.     

Expression of a soluble form of LFA-1 and demonstration of its binding activity with ICAM-1

The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell cycle-dependent fashion in vitro and appear to be regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization expression patterns of Brca1 and Brca2 mRNA in vivo in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues including testes, ovaries, and hormonally altered ovaries. Brca1 and Brca2 are expressed concordantly in proliferating cells of embryos, and the mammary gland undergoing morphogenesis and in most adult tissues. The expression pattern of Brca1 and Brca2 correlates with the localization of proliferating cell nuclear antigen, an indicator of proliferative activity. In the ovary, Brca1 and Brca2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles. In the testes, Brca1 and Brca2 were expressed in mitotic spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes revealed that the time course of Brca2 expression was delayed in spermatogonia relative to Brca1. Thus, while Brca1 and Brca2 share concordant cell-specific patterns of expression in most proliferating tissues, these observations suggest that they may have distinct roles during meiosis.     

Urinary and plasma urodilatin measured by a direct RIA using a highly specific antiserum

Urodilatin (95-126) (URO) appears to play a major physiologic role in fluid homeostasis and produces major changes when administered intravenously. Here we describe a monospecific, high-affinity antiserum against URO with no cross-reactivity (<0.01%) against the structural highly homologous atrial natriuretic peptide 99-126 (ANP-99-126), ANP analogs, and related peptides such as brain natriuretic peptide. A competitive RIA was developed, based on this antiserum. Urine samples with or without ethanol extraction and plasma samples without pretreatment were analyzed by the RIA, which had a detection limit of 10.5 ng/L, a linear measuring range between 10.5 and 1000 ng/L, and recoveries of 93-102% in urine and 90-104% in plasma. The intraassay CVs were 8.2% and 8.1% for urine samples with 269 and 669 ng/L URO; the interassay CV was 9.7% at 839 ng/L. Using this assay, we present URO data for urine from healthy volunteers receiving low and routine sodium diets and from clinical urine specimens; we also present pharmacokinetic data for URO in plasma from patients suffering from bronchial asthma and treated by URO infusion.     

Molecular properties and activity of a carboxyl-terminal truncated form of xylanase 3 from Aeromonas caviae W-61

Aeromonas caviae W-61 produces five species of xylanases, xylanases 1, 2, 3, 4, and 5 [Nguyen, V.D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993) and Appl. Environ. Microbiol., 57, 445-449 (1991)]. While preserving a purified xylanase 3 preparation from A. caviae in solution at 4 degrees C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one. It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease. We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing. By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr. Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan. In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products. Xylanase 3 hydrolysed X4 through X6. However, Xyn3tr had no activity towards X4 and X5. The recombinant Xyn3tr and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E. coli harboring the plasmids pXYN3 and pXYN3tr, which include xyn3 and xyn3tr genes, respectively, and their enzymatic activities were measured. The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr. Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from beta-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.     

Eel ventricular natriuretic peptide: isolation of a low molecular size form and characterization of plasma form by homologous radioimmunoassay

In order to determine the relation between myocardial fibrosis and (1) angiotensin converting enzyme (ACE) binding density, (2) receptor binding of ACE-related peptides, angiotensin II (AngII) and bradykinin (BK), and (3) the regulation of myocardial ACE by circulating Ang II, we used in vitro quantitative autoradiography to localize and assess ACE ([125I]351A), AngII receptor (125I[Sar1, IIe8]AngII), and BK receptor ([125I]Tyr8) binding densities in the rat myocardium. The experimental groups were (1) AngII (9 micrograms/h subcutaneously) or aldosterone (0.75 microgram/h subcutaneously in uninephrectomized rats on a high-sodium diet) administered by implanted minipump to chronically increase or decrease circulating AngII, respectively, (2) unoperated, untreated age- and sex-matched control rats, and (3) age- and sex-matched uninephrectomized control rats on a high-sodium diet. In the same heart studied at 2, 4, and 6 weeks of hormone treatment, serial sections were assessed for myocardial (hematoxylin and eosin) and fibrillar collagen (picrosirius red) morphology. The authors found that (1) marked ACE binding was coincident with sites of fibrous tissue that appeared in both ventricles as either a perivascular fibrosis of intramyocardial coronary arterioles or microscopic scarring in response to AngII or aldosterone infusion, (2) ACE binding was independent of circulating AngII, (3) BK, not AngII, receptor binding density was markedly increased at fibrous tissue sites, and (4) high-density ACE and BK receptor binding were anatomically coincident. Thus, in these experimental models, ACE is related to fibrous tissue formation where it may use BK, not angiotensin I, as a substrate, and its binding density is independent of circulating AngII.