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PubMed1979

Effects of diffusible products of peroxidation of rat liver microsomal lipids.

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.

Animals↗
PubMed1998

cDNA cloning of a novel autoantigen targeted by a minor subset of anti-centromere antibodies.

Using autoimmune serum from a patient with anti-centromere antibodies, we have identified and partially characterized a novel protein with a mol. wt of approximately 27 kD (hereafter referred to as p27). A cDNA expression library was screened with this serum, and two overlapping inserts were isolated among three positive clones other than clones corresponding to centromere protein (CENP)-B and CENP-C. Analysis of the sequence showed an open reading frame of approximately 0.6 kb encoding 199 amino acids with a predicted mol. wt of 21.5 kD. Immunoblotting analysis with bacterial recombinant p27 showed that approximately 2% of anti-centromere antibody-positive patients had autoantibodies to p27, whereas only one of 215 autoimmune patients without anti-centromere antibodies reacted with the recombinant. All five cases with anti-p27 antibodies, who were diagnosed as having scleroderma and/or Sjögren's syndrome, showed internal organ involvement. Although affinity-purified anti-p27 human or mouse polyclonal antibodies failed to stain any cellular structures in an immunofluorescence study, the potential association of anti-p27 with anti-centromere antibodies suggests that this novel autoantigen might play a role in mitosis.

Aged↗
PubMed2001

Pleomorphic adenomas of the major salivary glands: a study of the capsular form in relation to surgical management.

This was a retrospective study of 126 primary pleomorphic adenomas to correlate capsular characteristics with tumour histopathology in relation to current surgical debate (parotidectomy versus local excision). Capsular thickness was measured by micrometry and tumours classified into subtypes (1-4). Evidence of fine needle aspiration damage (needle tracks, infarction) was sought. Minimal changes were seen in eight tumours. Tumour growth features (bosselations, enveloping) were present in 57% and 33%, respectively, also microinvasion (42%) and tumour 'buds' (12%). Parotid lesions possessed thicker capsules than submandibular tumours. There was little correlation between capsular thickness and cellular structure. The significant exception was large (> 25 mm) hypocellular parotid tumours which had thinner capsules and could be vulnerable to operative rupture. In 110 standard operations (parotidectomy, submandibular gland excision), capsular exposure was evident in 81%. Field irrigation is recommended to lessen the risk of tumour seeding. This study reaffirms many elements of capsular weakness and suggests that parotidectomy is the operation of choice.

Adenoma, Pleomorphic↗
PubMed2003

Overproduction of a conserved domain of fission yeast and mammalian translation initiation factor eIF4G causes aberrant cell morphology and results in disruption of the localization of F-actin and the organization of microtubules.

BACKGROUND: The recruitment of mRNA for translation involves the assembly at the 5'cap of a complex of three initiation factors: the cap binding protein eIF4E, the ATP-dependent RNA helicase eIF4A and the scaffold protein eIF4G. eIF4G mediates the binding of this mRNA-protein complex to the 43S ribosomal preinitiation complex. There is growing recognition that the components of the translational apparatus interact functionally with cytoskeletal components. Here we report specific effects of the over-expression of human and fission yeast eIF4G domains on cell morphology in Schizosaccharomyces pombe. RESULTS: A single gene encoding fission yeast eIF4G was identified and demonstrated to be essential. We have over-expressed fragments corresponding to the conserved functional domains of eIF4G. At expression levels that did not disrupt rates of overall translation or protein accumulation, a fragment of S. pombe eIF4G, 4G-NOB, corresponding to the minimal region of human eIF4G required to support cap-independent mRNA recruitment, was found to impair cell proliferation in fission yeast. This resulted from defects in cytokinesis, and was associated with the disruption of both microtubules and actin microfilaments. The over-expressed fragment was itself localized to the cell ends, the nuclear periphery and the septum. CONCLUSIONS: This is the first demonstration of a link between a translation initiation factor and mechanisms controlling cell morphology. The data suggest a direct or indirect interaction between the functional domains of eIF4G and cellular structures involved in cytokinesis.

Actins↗
PubMed2003

Review of methods used to estimate non-milk extrinsic sugars.

BACKGROUND: Those sugars in foods, which are potentially damaging to dental health, were classified by the Committee on Medical Aspects of Food Policy (COMA) as non-milk extrinsic sugars (NMES). The NMES include sugars outside the cellular structure of a food, excluding the sugars naturally present in milk and milk products. The NMES should contribute no more than 10% of energy intake (Department of Health, 1991). A number of studies have been published where NMES content of foods has been estimated. The purpose of this study was to carry out a comprehensive literature review using a methodical search strategy in order to identify the different methods that have been used for NMES estimation. METHODS: Databases searched were MEDLINE, EMBASE, Health-CD and Health Management Information Consortium (HMIC) (as sources of UK government and other official publications). RESULTS: In total, 32 publications were found in which NMES values were reported and five different methods to estimate NMES were identified. No published method provided sufficient information to clearly differentiate between methods and inadequate detail was given to support replication of any of the methods. Of these five methods, The Ministry of Agriculture, Fisheries and Food (MAFF) have published three different descriptions of methods of NMES estimation used in UK national dietary surveys published since 1989. However, one method has been described consistently in the National Diet and Nutrition surveys published since 1994. CONCLUSIONS: A single, uniform approach to the estimation of NMES for application in nutritional surveys is essential for cross-comparison between surveys. The results show that there is a clear need for one standardized approach for the estimation of NMES in foods.

Animals↗
PubMed2003

Contrasting selection pressures on components of the Ras-mediated signal transduction pathway in Drosophila.

The molecular genetics of several signal transduction pathways is well characterized, providing an opportunity to address the nature of the population genetic forces acting on functionally related suites of pleiotropic regulatory genes. Signal transduction is the process by which signals are transmitted from the cell surface to the nucleus or other cellular structures. It plays a fundamental role in regulating a wide range of developmental and physiological processes, many of which are likely to be subject to buffering mechanisms. Here we infer that contrasting selection pressures act on six components of the Ras signal transduction pathway by comparing sequences obtained from 25 alleles of Drosophila melanogaster with one allele of the sibling species D. simulans. The three most upstream components of the cascade, Ras, Drk and polehole, experience strong purifying selection, as they show no fixed amino acid differences between the species and just a handful of rare replacement polymorphisms within D. melanogaster. This portion of the pathway is likely to act as a control point in signal transduction, because the more downstream components Dsor1, corkscrew and Ksr, each show several amino acid replacements between the species. Furthermore, Ksr is nearly monomorphic within D. melanogaster, and application of the HKA and McDonald and Kreitman tests indicate that this gene may have experienced a recent selective sweep, suggesting that modifiers of Ras kinase signalling are the most likely source of quantitative variation associated with this core regulatory pathway.

Animals↗
PubMed2000

A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily.

The purification to homogeneity of p16, a protein with an electrophoretic mobility compatible with an apparent molecular mass of 16 kDa, from nuclei of ungerminated pea embryonic axes is described. A cDNA clone of its gene, which was designated psp54, was also isolated. The psp54 cDNA contains an open reading frame coding for a 54.4-kDa polypeptide (p54). p16 corresponds to the C-terminal third of p54, although the mechanisms by which the primary polypeptide could be processed are not yet known. The sequence of p54 is 60% identical with that of the precursor of a sucrose-binding soybean protein, and, to a lesser extent (31-34%), it shares homology with some storage proteins. p16 is also 30% homologous with Nhp2p, a yeast nuclear protein. The psp54 gene, present in a single copy in pea genome, starts being expressed during seed desiccation. Soon after rehydration in seed germination, p54 mRNA disappears and is no longer detectable in vegetative tissues, except in response to hydric stress (exposure to abscisic acid, osmolites or desiccation). p16 can be recovered from nuclei cross-linked to histone H3, when the disulfide bridges that occur in vivo are preserved. On the other hand, p16 shares some properties with dehydrins, which are thought to protect cellular structures against desiccation. We propose that the possible precursor polypeptide p54 belongs to the vicilin superfamily, members of which play a variety of roles. The function of p16 may be related to the protection of chromatin structure against desiccation during seed development.

Amino Acid Sequence↗
PubMed2001

Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry.

HSP90 is one of the most abundant proteins in the cytosol of eukaryotic cells. HSP90 forms transient or stable complexes with several key proteins involved in signal transduction including protooncogenic protein kinases and nuclear receptors, it interacts with cellular structural elements such as actin-microfilament, tubulin-microtubule and intermediate filaments, and also exhibits conventional chaperone functions. This protein exists in two isoforms alpha-HSP90 and beta-HSP90, and it forms dimers which are crucial species for its biological activity. PAGE, ESI-MS and MALDI-MS were used to study HSP90 purified from pig brain. The two protein isoforms were clearly distinguished by ESI-MS, the alpha isoform being approximately six times more abundant than the beta isoform. ESI-MS in combination with lambda phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain alpha-HSP90, with the diphosphorylated form being the most abundant. For the beta isoform, the di-phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross-linking showed a high percentage of alpha-alpha homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained.

Animals↗
PubMed2000

Functional role for the class IX myosin myr5 in epithelial cell infection by Shigella flexneri.

Efficient control of Shigella-induced, rho-dependent cytoskeletal rearrangements seems to be required to shape the delicate cellular structures associated with bacterial invasion of epithelial cells. We therefore studied a class IX myosin and rho antagonist, the GTPase-activating protein (GAP) myr5, for a potential role in the bacterial entry process. We show that myr5 is recruited into bacterial entry spots. The recruitment pattern resembled that of rhoC or ezrin, but not rhoA, rac or CDC42, while in vitro GAP activity of myr5 was similar for rhoA, B or C. Analysis of myr5 mutants suggested that GTPase- or ATP-binding activites are not required for Shigella-induced recruitment of this atypical myosin to the bacterial entry site. Functional studies revealed a potential dual role of the myosin functions and the GAP module of myr5 for bacterial internalization.

Cytoskeletal Proteins↗
PubMed1999

T cell stimulating stratum corneum antigens: characterization by chromatography and electrophoresis indicates limited diversity.

As part of a search for T cell autoantigens in inflammatory skin diseases, we have demonstrated proteinase K sensitive, denaturation stable, T cell stimulatory material with antigenic properties in aqueous extracts of stratum corneum from normal human skin. Activity was also demonstrable in extracts of whole epidermis. A combination of preparative, analytical, and microbore reversed phase high performance liquid chromatography, chromatofocusing, and denaturing preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated limited structural diversity. Five components were separated, with Mr values from 5 to 18 kDa and apparent PI values from 4.5 to 10. Three components were purified to near homogeneity and showed molecular weights of 5, 13.5, and 18 kDa. Their potency was shown by the ability to induce stimulation indices of 20-89 with peripheral blood mononuclear cells and >500 with T cell lines. Use of inhibitors indicated that the active materials were not generated by the in vitro actions of proteases during extraction. The five partially purified components induced a time course of peripheral blood mononuclear cell proliferation compatible with the effects of antigen rather than superantigen. The 5 kDa component was rigorously bulk purified to yield a fraction that induced potent T cell activation but contained minimal detectable protein, a further indication of its biologic potency. Normal stratum corneum thus contains previously undescribed T cell antigens of high potency but limited structural diversity. The present data form a basis for determining their structure, cellular origin, and pathogenic relevance.

Amino Acid Sequence↗
PubMed2001

Effect of angiotensin-converting enzyme inhibition on renal filtration surface area in hypertensive rats.

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitor treatment leads to protective effects on the cellular structure of the glomerulus and the kidney. The aim of this study was to determine whether ACE inhibition increases renal filtration surface area in the spontaneously hypertensive rat (SHR). METHODS: SHR were treated with the ACE inhibitor perindopril at a high dose (3 mg/kg/day) or a low dose (0.1 mg/kg/day) during the period of hypertension development, from 7 to 14 weeks of age. Some animals were treated concomitantly with the bradykinin B2 receptor antagonist, S16118. Tail-cuff systolic blood pressure and body weights were measured twice weekly. At termination of treatment, glomerular number and volume, length, and surface area of glomerular capillaries and renal filtration surface area were estimated using unbiased stereological techniques. RESULTS: There were significant dose-related reductions in blood pressure with high- and low-dose perindopril treatment. Neither low- nor high-dose perindopril treatment had any effect on glomerular number or size or glomerular capillary length and surface area. Hence, there was no significant difference in total renal filtration surface area between any of the experimental groups (8721 +/- 610 mm2 in untreated SHR and 7879 +/- 338 mm2 and 8767 +/- 437 mm2 in the low and high dose perindopril-treated groups, respectively). Coadministration of the bradykinin antagonist did not affect any of the glomerular parameters. CONCLUSIONS: ACE inhibition during the period of hypertension development does not lead to an enhanced glomerular capillary growth or increases in total renal filtration surface area in this model.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2002

LR white post-embedding colloidal gold method to immunostain MBP, P0, NF and S100 in glutaraldehyde fixed peripheral nerve tissue.

A variety of immunocytochemical techniques are now widely used for the electron and light microscopic examination of biological samples. They are employed routinely for investigating the role of certain proteins in nervous tissue. Immunoelectron microscopic studies require the tissue to be fixed and embedded in a solid support, which may disrupt cellular structures and destroy crucial antigens. A technique of post-embedding with LR white resin has been developed, and it has been shown that certain antigens tolerate fixation with glutaraldehyde. In this study, we optimized a previous post-embedding method using low-water-miscible low-temperature embedding resin (LR white) to immunostain MBP, P0, NF and S100 proteins in peripheral nerves fixed with a relatively high concentration of glutaraldehyde found to be compatible with the morphology of normally compacted nerve fibers from humans and adult animals. The main difference in the procedures described here from previous ones is the elimination of vibratome sectioning, rendering this immunostaining technique more accessible to neuropathological laboratories using standard equipment for the ultrastructural study of peripheral nerves. It may prove of value for localization and quantification of these proteins in normal and pathological conditions.

Animals↗
PubMed2006

[Aging and biological rhythms in primates].

All living organisms exhibit rhythmic activities in a wide variety of endocrine and behavioural parameters. These biological rhythms are endogenously generated by a circadian clock, and they are entrained by cyclic variations of environmental factors called synchronizers. Aging is associated with changes in amplitude and temporal organization of many daily and seasonal rhythms. In humans, daily rhythms of sleep, thermoregulation and hormonal secretion are severely altered with aging. Except in humans, studies on primates are scarce. However, age-related effects on biological rhythms are relatively consistent among primate species studied to date, including humans. Therefore, non human primates are of valuable use for such investigations. Most studies have been performed on the Rhesus macaque (longevity 35-40 years) and on the gray mouse lemur (longevity 10-12 years). Like in humans, the rest-activity rhythm becomes fragmented in aged primates, and shows an increased activity during the resting period. Aging induces a decrease in amplitude of the body temperature rhythm and an increase in energy consumption. Various hormonal secretions exhibit a decrease with aging, but the rhythmic components of these declines have not always been depicted. Moreover, changes (amplitude or phase) in daily variations depended of the hormonal secretion tested. Taken together, these results suggest that the biological clock in the brain would be a primary target of aging. The main central clock is located in the suprachiasmatic nucleus of the hypothalamus whose endogenous oscillations are entrained by light. In this brain structure, cellular function and sensitivity to light show drastic changes with age in the mouse lemur. The precise knowledge of age-related alterations of biological rhythms in primates can have important consequences on the development of new treatments to maintain or restore biological rhythmicity in the elderly.

Aging↗
PubMed1997

Localization of rat pancreatitis-associated protein during bile salt-induced pancreatitis.

BACKGROUND & AIMS: Pancreatitis-associated protein (PAP), which is overexpressed in pancreatic acinar cells, appears in pancreatic juice and serum after acute pancreatitis. The aim of this study was to examine intracellular localization of PAP and amylase in healthy rat pancreas and pancreatitis pancreas to ascertain PAP transport from the rough endoplasmic reticulum to the zymogen granules into the acinar lumen. METHODS: Control rats and rats with taurocholate-induced pancreatitis were killed after 24 hours. Pancreata prepared for light and electron microscopy were used for amylase and PAP detection with specific antibodies. RESULTS: Induced acute pancreatitis disturbed the gross histology and ultrastructure of the acinar cells with the formation of new intracellular fibrous material into the cytoplasm, which was also found into the acinar lumen. PAP is almost absent from normal acinar cells; after acute pancreatitis, it appears in rough endoplasmic reticulum, it is strongly present in normal and abnormal zymogen granules, and it remains an important component of the fibrous material. Except for its exclusive presence in fibrous material, PAP is always colocalized with amylase in the other cell compartments. CONCLUSIONS: These observations show that the accumulated PAP into the acinar cells in response to acute pancreatitis behaves like all the other secretory proteins with the exception that it also accumulates in a new fibrillous cellular structure also found in the acinar lumen.

Acute-Phase Proteins↗
PubMed2004

Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo.

BACKGROUND & AIMS: A confocal laser endoscopy system has recently been developed that may allow subsurface imaging of living cells in colonic tissue in vivo. The aim of the present study was to assess its potential for prediction of histology during screening colonoscopy for colorectal cancer. METHODS: Twenty-seven patients underwent colonoscopy with the confocal endoscope using acriflavine hydrochloride or fluorescein sodium with blue laser illumination. Furthermore, 42 patients underwent colonoscopy with this system using fluorescein sodium. Standardized locations and circumscript lesions were examined by confocal imaging before taking biopsy specimens. Confocal images were graded according to cellular and vascular changes and correlated with conventional histology in a prospective and blinded fashion. RESULTS: Acriflavine hydrochloride and fluorescein sodium both yielded high-quality images. Whereas acriflavine hydrochloride strongly labeled the superficial epithelial cells, fluorescein sodium offered deeper imaging into the lamina propria. Fluorescein sodium was thus used for the prospective component of the study in which 13,020 confocal images from 390 different locations were compared with histologic data from 1038 biopsy specimens. Subsurface analysis during confocal laser endoscopy allowed detailed analysis of cellular structures. The presence of neoplastic changes could be predicted with high accuracy (sensitivity, 97.4%; specificity, 99.4%; accuracy, 99.2%). CONCLUSIONS: Confocal laser endoscopy is a novel diagnostic tool to analyze living cells during colonoscopy, thereby enabling virtual histology of neoplastic changes with high accuracy. These newly discovered diagnostic possibilities may be of crucial importance in clinical practice and lead to an optimized rapid diagnosis of neoplastic changes during ongoing colonoscopy.

Acriflavine↗
PubMed2002

Evidence of oncotic cell death and DNA fragmentation in human hypertrophic chondrocytes in chondro-osteophyte.

OBJECTIVE: To investigate the population and morphology of in situ terminal deoxynucleotidyle transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) stain positive non-apoptotic chondrocytes in hypertrophic zone of human chondro-osteophytes. MATERIALS AND METHODS: Chondro-osteophytes from osteoarthritic patients were obtained at joint replacement surgery. Apoptosis was verified by light microscopic examination of Safranin O stained sections and TUNEL stain. TUNEL staining was also performed on hydrophilic resin embedded semi-thin and ultra-thin sections combined with the treatment with streptavidin-gold conjugates, observed by light microscopy with silver enhancement technique (TUNEL-LM with SE) and transmission electron microscopy (TUNEL-TEM) respectively for the simultaneous evaluation of cellular structure and DNA fragmentation. RESULTS: In paraffin embedded sections (N=18), 31.5+/-6.1% of cells in the hypertrophic zone were TUNEL positive, but only 3.8+/-1.2% cells in this zone showed apoptotic appearances with cell shrinkage and nuclear condensation. Both in TUNEL-TEM and TUNEL-LM with SE, gold particles, which indicate DNA fragmentation, were observed within the nucleus of morphologically apoptotic chondrocytes, as well as of disintegrated, swollen chondrocytes. CONCLUSIONS: In human chondro-osteophytes, hypertrophic chondrocytes might die by oncotic cell death with DNA fragmentation, as well as apoptosis.

Adult↗
PubMed2002

The nucleotide: DNA sequencing and its clinical application.

Information determining cellular structure and function is contained in chromosomal DNA. Genes, regions of DNA encoding this information, are composed of specific sequences of nucleotides. DNA sequencing methods have been developed to identify these sequences. Even subtle alteration (or mutation) of these sequences can lead to many human syndromes and diseases. This article reviews 1) the structure of the nucleotide, 2) the methods of DNA sequencing, and 3) its recent clinical application in analysis of the nevoid basal cell carcinoma syndrome.

Basal Cell Nevus Syndrome↗
PubMed2000

Energy restriction dilutes the changes related to dietary fat type in membrane phospholipid fatty acid composition in rats.

To investigate liver cell membrane phospholipid (PL) fatty acid (FA) composition in response to the consumption of different types of dietary fat and graded levels of energy intake, rats were fed for 10 weeks on a diet containing either fish oil, safflower oil, or beef tallow. Within each dietary fat group, subgroups were either provided free access to food or energy-restricted to 85% or 70% of the ad libitum intake by reducing the dietary carbohydrate content while keeping other macronutrient intakes constant. Higher (P < .05) proportions of docosahexaenoic acid, linoleic acid, and monounsaturated FA were observed in the membrane PL of the fish oil, safflower oil, and beef tallow groups, respectively, resembling the FA composition in the diets. However, such modifications of dietary FA composition in, membrane PL FA were influenced by body energy status. The higher docosahexaenoic acid and total n-3 FA content in phosphatidylcholine (PC), sphingomyelin (SPH), and phosphatidylserine (PS) of the ad libitum fish oil group compared with the other dietary groups no longer existed when energy supply was restricted. Therefore, reducing energy intake tended to dilute the changes of membrane PL FA composition occurring as a function of dietary FA composition. These data suggest that the influence of dietary fat type on cellular structure and perhaps function becomes increasingly important with progressively positive energy balance.

Animals↗