Evaluation of correlated studies using liquid cell and cryo-transmission electron microscopy: Hydration of calcium sulphate and the phase transformation pathways of bassanite to gypsum.

Insight into the nucleation, growth and phase transformations of calcium sulphate could improve the performance of construction materials, reduce scaling in industrial processes and aid understanding of its formation in the natural environment. Recent studies have suggested that the calcium sulphate pseudo polymorph, gypsum (CaSO4 ·2H2 O) can form in aqueous solution via a bassanite (CaSO4 ·0.5H2 O) intermediate. Some in situ experimental work has also suggested that the transformation of bassanite to gypsum can occur through an oriented assembly mechanism. In this work, we have exploited liquid cell transmission electron microscopy (LCTEM) to study the transformation of bassanite to gypsum in an undersaturated aqueous solution of calcium sulphate. This was benchmarked against cryogenic TEM (cryo-TEM) studies to validate internally the data obtained from the two microscopy techniques. When coupled with Raman spectroscopy, the real-time data generated by LCTEM, and structural data obtained from cryo-TEM show that bassanite can transform to gypsum via more than one pathway, the predominant one being dissolution/reprecipitation. Comparisons between LCTEM and cryo-TEM also show that the transformation is slower within the confined region of the liquid cell as compared to a bulk solution. This work highlights the important role of a correlated microscopy approach for the study of dynamic processes such as crystallisation from solution if we are to extract true mechanistic understanding.

Fitting and flailing: recognition of paediatric antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a systemic autoimmune condition where the presence of antiphospholipid antibodies is thought to predispose to thrombotic events. It is uncommon in the paediatric population, but current diagnostic criteria are based on adult population studies, making assessment of its true paediatric prevalence difficult. We present two cases of paediatric APS, who presented with primary neurological events, and discuss approaches to diagnosis, interpretation of screening investigations, including antinuclear antibodies (ANA), anti-extractable nuclear antigen (ENA) antibodies and lupus anticoagulant. Possible approaches to the management of paediatric APS are discussed.

Calf stretching in non-weight bearing versus weight bearing.

Limited ankle dorsiflexion passive range of motion (DF PROM) has been associated with lower extremity overuse injuries. Therefore, clinicians often prescribe stretching exercises to increase ankle DF PROM. However, there is limited evidence to indicate if any particular gastrocnemius stretching exercise results in greater improvement in DF PROM. The aim of this study was to determine if gastrocnemius stretching in non-weight bearing (NWB) or weight bearing (WB) results in a greater increase of ankle DF PROM. 28 healthy volunteers, aged 18-55 years, who exhibited less than 10 degrees of ankle DF PROM completed the study. Participants were randomized into 2 stretching groups: NWB and WB. Both groups completed a 3-week home gastrocnemius stretching program, consisting of 5 repetitions held for 30 s each, 2 times daily. Participants' ankle DF PROM was measured with a blinded standard goniometer in NWB and WB positions before and after participation in a 3-week home gastrocnemius stretching program. Two 3-way mixed model ANOVAs demonstrated no significant difference in ankle DF PROM between the NWB and WB groups for either the NWB measurement condition (p=0.49) or WB measurement condition (p=0.86). Gastrocnemius stretching exercises performed in NWB or WB were equally effective in increasing ankle DF PROM.

Celiac disease (gluten-sensitive enteropathy).

Celiac disease may be initially detected in either children or adults, even in the elderly. This small intestinal mucosal disorder is gluten-dependent and causes nutrient malabsorption, often with diarrhea and weight loss. Diagnosis depends on detection of typical biopsy changes in the proximal small bowel along with an unequivocal response to a gluten-free diet. Recurrent changes usually result from poor adherence to the gluten-free diet, sometimes intentional, or from consumption of unsuspected gluten sources. In others, the original diagnosis may not be correct (e.g., duodenal involvement with Crohn's disease) or another cause for symptoms may have supervened (e.g., collagenous colitis, functional bowel disease). Rarely, a complication may occur (e.g., collagenous sprue, lymphoma). In some, the gluten-dependent nature of the small bowel disorder was not initially documented and biopsy changes continued despite a gluten-free diet. These have a sprue-like intestinal disorder, also labeled unclassified sprue. This represents a small, but likely, heterogeneous group, and in these, intractable symptoms may be present and, in some, lymphoma is eventually diagnosed.

Phase determination by multiple-wavelength x-ray diffraction: crystal structure of a basic "blue" copper protein from cucumbers.

A novel x-ray diffraction technique, multiple-wavelength anomalous dispersion (MAD) phasing, has been applied to the de novo determination of an unknown protein structure, that of the "blue" copper protein isolated from cucumber seedlings. This method makes use of crystallographic phases determined from measurements made at several wavelengths and has recently been made technically feasible through the use of intense, polychromatic synchrotron radiation together with accurate data collection from multiwire electronic area detectors. In contrast with all of the conventional methods of solving protein structures, which require either multiple isomorphous derivatives or coordinates of a similar structure for molecular replacement, this technique allows direct solution of the classical "phase problem" in x-ray crystallography. MAD phase assignment should be particularly useful for determining structures of small to medium-sized metalloproteins for which isomorphous derivatives are difficult or impossible to make. The structure of this particular protein provides new insights into the spectroscopic and redox properties of blue copper proteins, an important class of metalloproteins widely distributed in nature.

Relevance of segmental colitis with diverticulosis (SCAD) to other forms of inflammatory bowel disease.

A well localized inflammatory process involving only the sigmoid colonic segment associated with diverticulosis (SCAD), has become increasingly recognized as a distinct clinical and pathological disorder, usually described in older adults, often with rectal bleeding. Although some resolve spontaneously, most patients appear to respond to treatment only with 5-aminosalicylate. Endoscopic evaluation reveals a nonspecific inflammatory process localized in the sigmoid colon that usually completely resolves with histologically normal colonic mucosa. Recurrent symptoms with evidence of recurrent segmental colitis may occur, but most have an entirely benign clinical course. Further definition of the underlying molecular signalling that occurs in this apparently distinctive disorder may be critically important to understand the elements of a colonic inflammatory process that can completely and spontaneously resolve.

Beam-induced oxidation of mixed-valent Fe (oxyhydr)oxides (green rust) monitored by STEM-EELS.

Analytical transmission electron microscopy (TEM) is often used to investigate morphologies, crystal structures, chemical compositions and oxidation states of highly reactive mixed-valent mineral phases. Of prime interest, due to its potential role in toxic metal remediation, is green rust sulphate (GRSO4) an FeII-FeIII layered double hydroxide. In this study, we quantified the effects that TEM analysis has on GRSO4 in order to ensure the measured material properties are a result of synthesis and reaction kinetics, and not due to sample preparation and analysis technique. To do this, we compared two sample preparation techniques (anoxic drop-cast with drying, and frozen-hydrated cryogenic) and exposed samples to the electron beam for several minutes, acquiring fluence series between ca. 40 e- Å-2 and 10,000 e- Å-2. TEM imaging and electron diffraction showed that the hexagonal plate-like morphology and crystal structure of GRSO4 were largely unaffected by sample preparation and analysis technique. However, quantitative analysis of a series of monochromated Fe L3,2-edge electron energy loss spectra (EELS) showed that electron irradiation induces oxidation. We measured an Fe(II)/Fe(III) ratio of 1.94 (as expected for GRSO4) at 50 e- Å-2. However, above this fluence, the ratio logarithmically decreased and dropped to ca. 0.5 after 1000 e- Å-2. This trend was approximately the same for both sample preparation techniques implying that it is the beam alone which causes valence state changes, and not exposure to oxygen during transfer into the TEM or the vacuum of the TEM column. Ultimately this work demonstrates that GR valence can be quantified by EELS provided that the sample is not over exposed to electrons. This also opens the possibility of quantifying the effect of redox-sensitive toxic metals (e.g., As, Cr, Se) on Fe oxidation state in GR phases (relevant to the treatment of contaminated soils and water) with a higher spatial resolution than other techniques (e.g., Mössbauer spectroscopy).

Pearls and pitfalls in the diagnosis of adult celiac disease.

In adults with diarrhea or suspected malabsorption, a diagnosis of celiac disease requires that two criteria be fulfilled: first, a demonstration of typical pathological changes of untreated disease in biopsies from the proximal small bowel; and second, evidence should exist that clinical (and/or pathological) changes are gluten-dependent, most often as an unequivocal response to a gluten-free diet. Pathological abnormalities of celiac disease may include severe ('flat') or variably severe (mild or moderate) small bowel mucosal architectural abnormalities that are associated with both epithelial cell and lymphoid cell changes, including intraepithelial lymphocytosis. Architectural changes tend to be most severe in the duodenum and proximal jejunum and less severe, or absent, in the ileum. These findings, while characteristic of celiac disease, are not specific because several other conditions can produce similar changes. Some serological assays (eg, tissue transglutaminase antibody assays) are very useful screening tools in clinical practice because of their high specificity and sensitivity, but these do not provide a definitive diagnosis. The most critical step in the diagnosis of celiac disease is the demonstration of its gluten-dependent nature. The clinical response to gluten restriction in celiac disease is usually reflected in the resolution of diarrhea and weight gain. Normalization of biopsy changes can be first shown in the most distal intestinal sites of involvement, and later, sometimes only after prolonged periods (months to years) in the duodenum. Rarely, recurrent (or refractory) celiac disease may occur after an initial gluten-free diet response. Finally, some with 'sprue-like intestinal disease' cannot be classified because a diet response fails to occur. This may be a heterogeneous group, although some are eventually found to have a malignant lymphoma.

Intestinal assimilation of a proline-containing tetrapeptide. Role of a brush border membrane postproline dipeptidyl aminopeptidase IV.

The mechanism of hydrolysis and absorption of a proline-containing tetrapeptide, Leu-Pro-Gly-Gly (10 mM) by rat intestine was examined in vivo by using jejunal perfusion methods. The peptide substrate and hydrolysis products were analyzed by use of an automated amino acid analyzer. Leucine, proline, and glycine were absorbed by the intestine at a significantly higher rate from the tetrapeptide than from an equivalent amino acid mixture. The analysis of the hydrolytic products in the lumen during in vivo perfusion of the tetrapeptide showed that two dipeptides, Leu-Pro and Gly-Gly, were the major products. These two dipeptides were also the major hydrolytic products when a purified rat intestinal brush border membrane preparation was incubated with Leu-Pro-Gly-Gly. The rate of hydrolysis of the tetrapeptide was much higher than that for several other proline-containing peptides (Leu-Pro, Pro-Leu, and Pro-Gly-Gly) that were tested. Studies using Gly-Pro-beta-naphthylamide, a specific substrate for postproline dipeptidyl aminopeptidase IV, showed that this enzyme is mainly localized to the brush border membrane and is responsible for the hydrolysis of the tetrapeptide into the two dipeptides Leu-Pro and Gly-Gly. Thus, brush border membrane dipeptidyl aminopeptidase IV very likely plays an important role at the intestinal mucosal cell surface in the final stages of digestion of proline-containing peptides.

Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex.

Mast cells permeabilized by treatment with streptolysin-O in the presence of Ca2+ and GTP-gamma-S can secrete almost 100% of their contained N-acetyl-beta-D-glucosaminidase. If these stimuli are provided to the permeabilized cells after a delay, the response is diminished and the ability of the cells to undergo secretion runs down progressively over a period of about 30 min. This is thought to be due to the loss of key proteins involved in the exocytotic mechanism. Using this effect as the basis of a biological assay, we have isolated a protein from bovine brain cytosol that retards the loss of responsiveness to stimulation by Ca2+ and GTP-gamma-S. Purification of this protein and peptide sequencing have enabled us to identify it as the small GTP-binding protein rac complexed to the guanine nucleotide exchange inhibitor rhoGDI. Both proteins are required to retard the loss of the secretory response, while purified rhoGDI applied alone accelerates the rundown.

Atypical experiences of captive chimpanzees (Pan troglodytes) are associated with higher hair cortisol concentrations as adults.

Experiences during early development are influential on the lives of human and non-human primates into adulthood. The population of captive chimpanzees in the USA can provide insight into this relationship, as collectively they have experienced a wide range of exposure to both conspecifics (those raised in natal groups) and humans (those raised as personal pets or performers). Our study investigated chimpanzee exposure to humans using a continuous measure of categorization, the chimpanzee-human interaction index, and the relationship between this experience and cortisol concentrations in adulthood. Historical records and hair samples were collected from 60 chimpanzees which were socially housed in 13 zoos and sanctuaries. We found that more human exposure throughout the life of a chimpanzee was associated with higher hair cortisol concentrations in adulthood. Sex was also a significant factor affecting cortisol concentration, with male chimpanzees having higher cortisol concentrations than female chimpanzees. These results build upon the extensive literature about aversive effects of atypical social histories for chimpanzees and emphasize to managers the importance of monitoring potential negative health consequences and social deficits these individuals may exhibit.

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution.

BACKGROUND: Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported. RESULTS: The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains. The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement. All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A. CONCLUSIONS: There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Effects of differing purified cellulose, pectin, and hemicellulose fiber diets on fecal enzymes in 1,2-dimethylhydrazine-induced rat colon carcinogenesis.

The fecal microflora enzymes, beta-glucuronidase and beta-glucosidase, as well as fecal bacterial counts, were examined during colon carcinogenesis in rats administered parenteral 1,2-dimethylhydrazine and fed nutritionally equivalent diets free of fiber or containing one of three single sources of dietary fiber (cellulose, hemicellulose, and pectin). Whereas pectin-fed animals had increased fecal beta-glucuronidase activities, those fed cellulose and hemicellulose, two fibers protective in dimethylhydrazine colon neoplasia, had decreased activities. Although fecal bacterial counts were not significantly changed, similar differential changes in fecal beta-glucosidase activity were noted: cellulose but not pectin or hemicellulose feeding was associated with reduced activity. Although cellulose fiber may cause differing physiological effects resulting in a reduction in colonic neoplasia development in this experimental animal model, decreased bacterial metabolic enzyme activation of carcinogens or cocarcinogens may lead to diminished exposure of colonic cells to exogenous or endogenous mutagens.

Differential effects of surgical suture materials in 1,2-dimethylhydrazine-induced rat intestinal neoplasia.

The incidence, distribution, size, and histopathology of rat small and large bowel tumors induced by sequential administration of 1,2-dimethylhydrazine followed by cecal placement of one of six differing types of suture materials were systematically examined. In addition, measurements of beta-glucuronidase activities in large bowel contents followed by fecal trace metal determinations were done. The results indicate that specific slowly absorbed and nonabsorbable suture materials in the absence of a surgical anastomosis promote tumor induction locally in the rat cecum. In addition, cecal suture material composed of multifilament stainless steel wire enhanced tumor development at a "downstream" site in the distal colon, paralleling increased fecal beta-glucuronidase activities at this site and implicating a possible luminally mediated mechanism for colon tumor development in this animal model.

Use of unscheduled DNA synthesis in freshly isolated human intestinal mucosal cells for carcinogen detection.

Mucosal cells freshly isolated from human intestine with pronase retain the capacity to undergo DNA repair synthesis (unscheduled DNA synthesis) during a 2-hr exposure to the carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine, and the procarcinogen, aflatoxin B1. This procedure combining the use of human intestinal mucosal cells and the measurement of unscheduled DNA synthesis may provide a highly relevant and convenient test system for the detection of cell-specific, direct-acting, and activation-dependent chemical carcinogens. The use of whole-cell preparations in such in vitro studies may be of additional significance in view of growing evidence for artefactual metabolism by subcellular fractions.

A double-blind study on the effect of purified cellulose dietary fiber on 1,2-dimethylhydrazine-induced rat colonic neoplasia.

The incidence, distribution, size, and histopathology of grossly visible colonic tumors induced by parenteral administration of 1,2-dimethylhydrazine were examined in rats fed either a chemically defined fiber-free diet or a nutritionally and calorically equivalent diet containing a purified fiber component, microcrystalline cellulose. This double-blind study indicates that cellulose ingestion was associated with reduced numbers of animals involved with colonic neoplasia as well as a reduction in the total numbers of colonic tumors. Furthermore, this protective effect of cellulose appears to be time dependent and associated with a shift in tumor distribution from the proximal colon to a more distal site. Cellulose fiber had no apparent effect on colonic tumor size, histopathology, or the incidence of other tumors known to occur in this experimental animal model. This study strongly supports the hypothesis that fiber is an important protective agent against colonic neoplasia development. While the mechanism for this protective effect remains obscure, it appears to be temporally related to the duration of fiber ingestion as well as to a differential fiber effect on either the luminal content or the mucosa of the proximal and distal colon.

A double-blind study on the effects of differing purified cellulose and pectin fiber diets on 1,2-dimethylhydrazine-induced rat colonic neoplasia.

The incidence, distribution, size, and histopathology of colonic tumors induced by parenteral administration of 1,2-dimethylhydrazine were examined in rats fed a chemically defined fiber-free diet or nutritionally and calorically equivalent diets containing either 4.5 or 9.0% purified cellulose or pectin. This double-blind study indicates that cellulose is protective against experimental colonic neoplasia. Although the precise mechanism for this protective effect remains to be elucidated, it was not cellulose dose dependent and appeared to depend on administration during injection of carcinogen. Furthermore, this study provides strong evidence that identical amounts of cellulose and pectin fed as the sole source of fiber in chemically defined diets exert strikingly different effects in relation to development of intestinal neoplasia in this animal model.

beta-Hexosaminidase isoenzymes in tissues, cultured cells, and media from human fetal intestine and colonic adenocarcinoma.

Isoelectric focusing of tissue homogenates revealed a predominance of beta-hexosaminidase B in colonic adenocarcinoma, whereas beta-hexosaminidase A was greater in paired normal-appearing colonic mucosa from the same patients as well as in a specimen of human fetal colonic mucosa. Because of the recognized cellular heterogeneity of these tissues, our studies were extended to an examination of these isoenzymes in 20 cultured epithelial cell lines derived from human fetal intestine and colonic carcinoma as well as the secreted enzymes in their culture media. While the B:A isoenzyme ratio was higher in human cancer cells as compared to fetal cells, some of the cancer cell lines had a greater proportion of the A isoenzyme. Examination of the isoenzyme profiles in the media of these cells revealed a greater B:A ratio whether of fetal or cancer cell origin. These studies parallel the apparent biological differences of neoplastic colonic epithelium occurring in vivo and are reminiscent of reported oncodevelopmental changes in enzymatic properties present in some malignant tissues. The differential stabilities of these two isoenzymes derived from the culture media of both types of cell lines in vitro may limit their value as markers of human colonic neoplasia.

An NMR investigation of electron transfer in the copper-protein, plastocyanin.

1. The proton NMR spectra of oxidised and reduced French bean plastocyanin have been recorded on a 270 MHz pulsed spctrometer. 2. The spectrum of a mixture containing the protein in the paramagnetic Cu(II) and diamagnetic Cu(I) states is a superposition of the separate spectra. When ferrirate spectra. 3. The results show that self-exchange between Cu(II)- and Cu(I)-plastocyanin is slow on the NMR time scale (kex less than 2-10(4) M-1-s-1 at 50 degrees C), and that electron transfer in the presence of ferricyanide is rapid (k greater than 1-10(5) M-1-s-1).