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PubMed2006

Chlamydophila pneumoniae and human cytomegalovirus in atherosclerotic carotid plaques--combined presence and possible interactions.

The aim of our study was to investigate the combination of Chlamydophila pneumoniae and human cytomegalovirus (HCMV) as a pathogenic factor in atherosclerosis. Accordingly, we tested by means of PCR and immunohistochemistry the presence of these pathogens in the same atherosclerotic carotid specimen. The histology of the samples and the patients' antibodies against these pathogens were evaluated. Further, we examined the impact of C. pneumoniae and HCMV infection on the gene expression of the human monocytic cell line U937. Six of the 22 samples contained only C. pneumoniae, 4 contained only HCMV, 7 contained both C. pneumoniae DNA and/or antigens of both pathogens, and 5 samples were negative. No correlation was found between the presence of these microbes and either the cellular structure of the plaques, or the serostatus of the patients. The infection of U937 cells with HCMV and especially C. pneumoniae induced inflammation and atherosclerosis-related genes. Furthermore, the doubly-infected cells produced higher levels of the mRNA of pro-platelet basic protein and fatty acid binding protein 4. In conclusion, C. pneumoniae is often present in combination with HCMV in atherosclerotic carotid lesions. The in vitro coinfection model reveals that the doubly-infected monocytes are potent expressors of proatherosclerotic genes, suggesting that this coinfected population may accelerate the process of atherosclerosis.

Aged↗
PubMed1986

[Cytologic examination of the cerebrospinal fluid in the diagnosis of primary or metastatic neoplasms of the nervous system].

The authors report 6 cases of cerebrospinal fluid (CSF) examinations in which malignant cells were found. In 5 cases the finding was incidental: medulloblastoma (case 1); malignant melanoma (case 2); adenocarcinoma (case 3); meningitis carcinomatosa (case 4) and neuroleukaemia (case 5). In only one case neuroleukaemia was suspected before the study of the CSF (case 6). The unexpected occurrence of malignant cells in the CSF demands the pathologist to be well acquainted with tumor cell cytology, in order to identify them providing so useful information that can decidedly influence subsequent diagnostic and therapeutic procedures. The cell collection technic developed in the authors' laboratory was considered adequate, because both inflammatory and malignant cells were securely identified. Its principle is to obtain thin air-dried smears that dry almost instantaneously on slides, in order the cellular structure be preserved. After centrifuging about 5-8 ml of CSF the tube is inverted so as to pour off the whole of the supernatant fluid. It is kept inverted with the operator's left hand, so that no drop of fluid can run back on to the cells. With his right hand the operator touches the bottom of the inverted tube with a Pasteur micropipette preciously made by distending a microhematocrit tube under the flame of a Bunsen burner and attached with adhesive tape to the handle of a wire loop 2 mm in diameter.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1986

The size and number of Langerhans islets correlated with their endocrine function: a morphometry on immunostained serial sections of adult human pancreases.

Surgical and autopsy specimens of normal pancreas from three human adults were submitted to morphometry of Langerhans islets and so-called extra insular islet cells, by measuring the islet radii on serial sections immunostained for either insulin or glucagon. All the islets contained in a test volume were measured including single cells, and the distribution of their radial sizes was studied. It was found that the distribution of islets was strongly skewed and continuous, with the majority of islets having radial values near to the minimum. From the fact that only one peak was found in the distribution curve, it is concluded that in spite of the distinction previously made between islets of Langerhans and so-called extra-islet cells, they are in fact both part of a continuous distribution. Moreover, the Weibull function, assumed as the theoretical distribution, fit well with the empirical histograms, giving support for the contention of continuous distribution with a single peak. Although the small islets account for a majority of the number of islets, they account for only a very small percentage of the islet volume. This is thought to indicate that the bulk of the endocrine functions of the pancreas are carried out by large islets, whereas the so-called extra-islet cells are likely to be their precursors. With regard to the cellular structure of the islets, it was found that the smaller the islet, the smaller the percentage of islets containing A cells, with the vast majority of cells in the smallest islets (often single cells) being B cells.

Adult↗
PubMed2003

Measurement of prompt DNA double-strand breaks in mammalian cells without including heat-labile sites: results for cells deficient in nonhomologous end joining.

Ionizing radiation induces prompt single-strand breaks and double-strand breaks in DNA. In addition, labile sites are induced that can be converted to breaks by heat or mild alkali. When such labile lesions are present within multiply damaged sites, additional double-strand breaks can form. Current protocols for measurement of DNA double-strand breaks involve a lysis step at an elevated temperature, and consequently breaks from heat-labile sites will be generated during lysis and will be included in the measurement. However, such sites may not develop into breaks within the cell and therefore may not need DNA double-strand break repair processes for elimination. We present here a new lysis and pulsed-field gel electrophoresis protocol that is carried out entirely at 0-4 degrees C and thus avoids inclusion of heat-labile sites in the measurement. The new recommended lysis procedure involves two steps: The first step includes proteinase K, which has sufficient activity at 0 degrees C to support lysis, and the second step includes a high-salt buffer to further free the DNA from proteins and other cellular structures. Using various tests, we conclude that lysis is sufficient with this procedure to allow accurate determination of double-strand breaks by pulsed-field gel electrophoresis. Using the new protocol, it was found that heat-labile sites account for 30% of the initial number of double-strand breaks measured by conventional protocols after exposure to low-LET radiation. In addition, we show that heat-labile sites that can be converted to double-strand breaks are repaired with fast kinetics and are almost completely eliminated after 1 h at 37 degrees C. A study of cells deficient in nonhomologous end joining reveals that the residual fast repair response typically seen in such cells is solely due to repair at heat-labile sites and is not due to repair of prompt DSBs.

Agar↗
PubMed2000

The glomerular slit diaphragm is a modified adherens junction.

The glomerular slit diaphragm between podocyte foot processes shares typical morphologic features with an adherens junction. Differentiated cultured podocytes form cellular structures comparable to filtration slits in vivo. At those sites, zonula occludens-1 (ZO-1) was coexpressed with P-cadherin as well as with alpha-, beta-, and gamma-catenin. In situ, P-cadherin was detected at the slit diaphragm in association with ZO-1 as shown by confocal microscopy and immunogold double labeling electron microscopy. P-cadherin expression in vivo and in vitro was confirmed by reverse transcription-PCR. These findings led to the concept that the slit diaphragm represents an adherens junction composed of P-cadherin, alpha-, beta-, and gamma-catenin, and ZO-1. In contrast to an adherens junction of a similar composition recently described in cultured fibroblasts, the slit diaphragm complex does not contain vinculin, which was found in nearby focal contacts. A P-cadherin-based adherens junction is well-suited to explain the zipper-like structure of the slit diaphragm. The present study should allow new avenues leading to the identification of additional slit diaphragm-associated proteins conferring specificity to this unique cell junction.

Animals↗
PubMed1990

Effects of diuretic treatment and of dietary sodium on renal binding of 3H-metolazone.

We report a series of experiments designed to determine if agents and conditions that have been reported to alter sodium reabsorption, Na-K-ATPase activity or cellular structure in the rat distal nephron might also regulate the density or affinity of binding of 3H-metolazone to the putative thiazide receptor in the distal nephron. Experimental conditions selected for study were acute (60-min) and chronic hydrochlorothiazide (HCTZ), acute acetazolamide, acute and chronic furosemide, and 14 days of varied intake of dietary sodium. The density of the binding of 3H-metolazone was increased 47% by acute HCTZ (P less than 0.001) and 39% (P less than 0.001) by acute furosemide. In contrast, acute acetazolamide produced no change in binding despite eliciting a dramatic diuresis. Chronic HCTZ (5 days) and chronic furosemide (7 days) increased binding of 3H-metolazone by 46% (P less than 0.001) and by 101% (P less than 0.001), respectively. Variation of dietary sodium intake over a range that allowed normal growth of the animal and that produced urinary excretion of Na varying from 0.28 to 2.62 mEq/100 g/day failed to alter the density of binding of 3H-metolazone. These studies are the first indication that the density of the thiazide receptor is regulated by a variety of both acute and chronic conditions that have previously been associated with changes in transport, ultrastructure or Na-K-ATPase activity in the distal nephron.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetazolamide↗
PubMed1984

The effectiveness of regenerating versus mature marrow in physiologic autogenous transplants.

Differences in rates of bone healing in animals and in human extraction wounds suggest that marrow tissue is more likely to form bone when transplanted at the onset of its most active regenerative period rather than at its maturity. To test this hypothesis, small segments of polyvinyl sponge were implanted into experimentally produced femur cavities in Sprague-Dawley rats, to provide matrices for regenerating cellular structure in the grafting procedures. Bone resulted in 16 of 22 cases (72.7%) in which regenerating marrow was transferred to the anterior eye chamber, compared with 4 of 23 cases (17.4%) in which mature marrow was used (P = 0.0002). No bone resulted when washed, polyvinyl sponge samples were implanted as controls in 6 cases. These findings indicate that with existing techniques it is probably more effective to transplant regenerating marrow than mature marrow.

Animals↗
PubMed2001

Human gingival fibroblast cytoskeleton is a target for volatile smoke components.

BACKGROUND: Several in vitro investigations have indicated that the particulate phase of cigarette smoke as nicotine affects many cell types including gingival fibroblasts, but few studies have examined the effect of volatile fraction on cellular structures involved in cell functions such as adhesion and proliferation. Since gingival fibroblast survival and reproduction are fundamental to maintaining the oral connective tissue as well as to wound healing, the effects of acrolein and acetaldehyde, volatile fractions of cigarette smoke, on cytoskeleton were examined in human gingival fibroblasts (HGFs) in vitro. METHODS: Human gingival fibroblast (HGF) strains from healthy subjects with non-inflamed gingiva were utilized. The cells were incubated in different concentrations of acrolein and acetaldehyde. Cell adhesion was evaluated after 3 hours. The influence of both substances on cytoskeletal structures, tubulin and vimentin intermediate filaments (VIF), was investigated using indirect immunofluorescence technique. RESULTS: The results show that both substances produced similar effects, resulting in a dose-dependent inhibition of HGF adhesion. Disturbance of HGF cytoskeleton consisted of a disruption of microtubules and vimentin microfilaments with alterations in cell shape. CONCLUSIONS: Our experimental findings suggest that volatile fractions of cigarette smoke such as acrolein and acetaldehyde, because their ability to bind and interact with the cytoskeleton, prevent HGF adhesion. Consequently the maintenance of the oral connective tissue and integrity and remodeling could be impaired. According to our morphological evidence, these findings confirm other clinical and epidemiological investigations reporting that volatile components of cigarette smoke could lead to the initiation and progression of periodontal disease.

Acetaldehyde↗
PubMed1996

Transfection maximizer increases the efficiency of calcium phosphate transfections with mammalian cells.

A variety of methods have been described for introducing DNA into cultured mammalian cells. Among these, calcium phosphate (CaPO4)-mediated transfection has gained wide acceptance due to the procedure's low cost, simplicity and suitability for a wide range of cell types. However, this method is limited by relatively low transfection efficiencies and poor reproducibility. To improve the utility of CaPO4 transfections, we have identified a transfection enhancer (CalPhos Maximizer) that dramatically increases the efficiency of CaPO4 transfections with mammalian cells. The CalPhos Maximizer is a cellular structural protein that improved CaPO4 transfections with all mammalian cell lines tested and yielded greater than 70% transfection efficiency in BHK-21 cells. In the presence of the CalPhos Maximizer, transfection times were decreased to as little as 2 h with no loss in performance. Because of the high transfection efficiency provided by the CalPhos Maximizer transfection system, gene expression can be assayed following overnight incubations, rather than the typical 48-72-h intervals required by conventional transfection methods. Moreover, the Maximizer reagent is compatible with, and improves the performance of, commercially available as well as home-made CaPO4 transfection systems.

Animals↗
PubMed2004

CD26/dipeptidyl peptidase IV: a regulator of immune function and a potential molecular target for therapy.

CD26 is a 110 kDa surface-bound ectopeptidase with intrinsic dipeptidyl peptidase IV (DPP IV) activity, which has multiple biological functions. In this review, we will focus specifically on work demonstrating that CD26 has a key role in immune function as a T cell activation molecule and a regulator of the functional effect of selected biological factors through its DPP IV enzyme activity. As further evidence of the important role played by this multifaceted molecule in immune regulation, we will also discuss experimental attempts from our laboratory and others to influence immune-mediated conditions through CD26 monoclonal antibodies and DPP IV activity with various agents, including anti-CD26 monoclonal antibodies and DPP IV chemical inhibitors. Of special significance from a clinical perspective is also CD26 effect on glucose metabolism through its DPP IV activity and its potential role as a therapeutic target in diabetes. In addition, we will review recent studies that describe the physical and functional interaction of CD26 with other essential cellular structures and the biological consequences of their association. In particular, we will present recent data from our laboratory that demonstrates the correlation between CD26, especially its DPP IV activity, and the key nuclear protein topoisomerase II alpha, an interaction that has important clinical implications. In summary, we will examine the biology of the multifaceted CD26/DPP IV molecule, focusing particularly on its function in immune regulation and its potential role as a molecular target for novel treatment modalities for a number of disease states, ranging from autoimmune diseases, diabetes to malignancies.

Animals↗
PubMed2003

Survivin: role in normal cells and in pathological conditions.

Survivin, an inhibitor of apoptosis (IAP) containing one baculovirus IAP repeat (BIR) domain, has been reported to be capable of regulating both cellular proliferation and apoptotic cell death. Survivin splice variants, survivin-DeltaEx3 and survivin-2B, have apparently retained and lost anti-apoptotic potential, respectively. As survivin was first discovered due to its high homology with effector cell protease receptor (EPR-1), a protein involved in blood coagulation, it has been suggested (but not proven) that EPR-1 may act as a natural anti-sense to survivin in cells. Survivin homologs have been found in non-human species. Survivin expression has been described during embryonic development and in adult cancerous tissues, with greatly reduced expression in adult normal differentiated tissues, particularly if their proliferation index is low. Survivin has been defined as a universal tumor antigen and as the fourth most significant transcriptosome expressed in human tumors. Although survivin is usually located in the cell cytoplasmic region and associated with poor prognosis in cancer, nuclear localisation, indicative of favorable prognosis, has also been reported. Survivin expression has also been reported in a number of proliferating normal adult tissues. Extensive research has been conducted, aimed at increasing our understanding of survivin, by determining its sub-cellular structure and location, mechanism(s) of action and control of expression. While much important information on this molecule has been accumulated, there are still many areas of controversy or limited information. Further research may enable exploitation of survivin overexpression in cancer compared to normal tissues, making survivin a potentially attractive target for cancer therapeutics.

Amino Acid Sequence↗
PubMed2005

Fibrinolysis at the fluid-solid interface of thrombi.

Thrombolysis is conventionally regarded as dissolution of the fibrin matrix of thrombi by plasmin, a protease generated by plasminogen activators from its inactive precursor, plasminogen. Typically plasminogen activation occurs on the surface of the clot, where fibrin behaves as a cofactor in this process, and plasmin also initiates its proteolytic action at the fluid-solid interface. Although the basic reactions of the plasminogen/plasmin system in fluid phase are well characterized in terms of classical enzymology, they cannot explain completely the interfacial fibrinolytic events. Recently new methods have been introduced for quantitative evaluation of plasminogen activation on gel-phase fibrin and heterogenous-phase proteolysis, an overview of the new methodology is presented. Following formation of an interfacial lytic zone, fibrin dissolution proceeds through propagation of this zone to the core of the clot, which depends on diffusion and permeation phenomena affected by the composition of thrombi. Phospholipids (originating from platelets) form a diffusion barrier to the thrombolytic agents and also bind some of them; structural cellular proteins (namely myosin) interact with the fibrin fibers masking their cofactor and plasmin-cleavage sites. The contribution of these recent findings to our understanding of the limitations of current thrombolytic therapy is discussed. Finally, attention is focused on the termination of thrombus-associated proteolytic action in an environment abundant in proteinase inhibitors. Thus, combining together the interfacial events in the initiation, progress and termination of thrombolysis, a concept for modeling the thrombus as a temporary fibrinolytic compartment is presented.

Fibrin↗
PubMed1998

Microorganisms and plant of Autonomous Biological Systems (ABS) samples.

Distribution of microorganisms and cellular structure of an Autonomous Biological Systems (ABS) were studied with a special attention to the effect of space environments. Viable cell densities measured by the direct fluorescence microscopic method were in the order of 10(5) cells/ml for fractions 1 (upper suspension) and 2 (lower suspension), and 10(6) cells/ml for fraction 3 (sediments). These values were 10 to 100 times larger than the values obtained by the classical colony forming unit method. No difference between flight and ground samples was observed in the vertical distribution of viable microorganisms when fractionation and analysis were carried out after recovery. Intracellular distribution of chloroplasts in higher green plants, Ceratophyllum demersum, of flight samples was disturbed after 10 days of flight (24hrs/day light on). After 4 months of flight (Mir/STS-79/81) with 24 hrs light on, Ceratophyllum demersum was completely disintegrated. On the other hand, in the second 4-months-flight experiment with 16 hrs/day light on, Ceratophyllum demersum was only slightly deteriorated.

Aquaculture↗
PubMedDate not supplied

[Catecholamines in the human diencephalon].

INTRODUCTION: Catecholamines are chemical compounds which play an important role as neurotransmitters in many vital functions of the organism. The paper presents a short survey of their biosynthesis, disintegration andfunctions, with respect to the neuroanatomical location of cell groups which contain these compounds. CATECHOLAMINES IN THE HUMAN HYPOTHALAMUS: Because the authors were most interested in the behavior of catecholamines in the diencephalon, particularly in the hypothalamus, they focussed their attention on cells secreting catecholamines (dopamine, noradrenaline, adrenaline, octopamine). The paper also deals with the connections between cellular structures which emit and receive the neuronal impulses that transport catecholamines as neurotransmitters. These include the following dopaminergic systems: nigrostriatal, tuberohypophysial, retinal, periventricular, periglomerular and dopaminergic systems in mesolimbic, mesocortical and diencephalic regions. The paper also indicates other areas in human brain with adrenaline and noradrenaline secreting cells.

Catecholamines↗
PubMed2026

Longitudinal Repeated Protein Measurements in a Multiethnic Cohort Identify Novel Diabetes Biomarkers That Reveal Unique Disease Pathways.

There is up to a fourfold increase in diabetes biomarkers identified with longitudinal repeated versus single time point proteomic measurements. The increase in biomarkers identified with longitudinal repeated measurements is supported by a similar proportion being nominated as causal for type 2 diabetes with Mendelian randomization. Proteins unique to the longitudinal repeated analyses highlighted biological pathways (e.g., posttranslational protein modification and cellular structure and cycle regulation) that were distinct from pathways enriched among the shared proteins (e.g., small-molecule metabolic and catabolic processes). Longitudinal protein measurements identify additional novel disease biomarkers and disparate biological pathways compared with single measurement analyses.

Journal Article↗
PubMedDate not supplied

Preoperative sagittal CT evaluation of the frontal recess.

Endoscopic surgical approaches for chronic frontal sinusitis require the reestablishment of adequate frontal sinus ventilation and drainage for relief of symptoms. After the resection of anterior ethmoid mucosal disease and cellular structure, the anterior to posterior depth of the nasofrontal beak to the base of skull at the insertion of the ethmoidal bulla (frontal sinus ostium) often represents a critical margin for functional success. However, little information concerning this dimension is available. Depending on intraoperative surgical judgment of this distance, extended endoscopic surgical procedures involving additional bone resection may be indicated. These approaches may be hazardous due to the proximity of the cranial cavity and orbit. In addition, secondary stenosis can result from the subsequent inflammatory response. Improved CT imaging, high resolution sagittal reformatting, and computer workstations provide the ability to obtain direct preoperative measurements of the frontal recess. We used a paramedian sagittal section and recorded the maximal anterior to posterior depth from the nasofrontal beak to the base of skull at the insertion of the ethmoidal bulla in 20 patients, 31 sides, undergoing primary endoscopic frontoethmoidectomy. In addition, we found a positive correlation between this distance and agger nasi air cell size measured in the same 31 sides.

Chronic Disease↗
PubMed1991

The histologic characteristics of the core of the fissula ante fenestram.

In earlier studies of perilymphatic fistulae, we described their histologic criteria and the clinical criteria for the identification and prediction of patencies of the labyrinth capsule related to disorders of hearing and balance. These patencies were either of the fissula ante fenestram or of the round window niche-posterior canal ampulla fissure. The permeability of the fibrous core of a patent fissula ante fenestram has been questioned, authors of some earlier articles arguing that the tissue is loose, others that it is dense. This study demonstrates loose fibrous and cellular structure in the fissula's tract, dense collagen at the circumference. The loose arrangement of tissue suggests that fluid can pass through this patency, and it appears similar to that of the spiral ligament which has been shown to contain perilymph by way of intercellular communications with the perilymphatic space.

Ear, Middle↗
PubMed1989

Transport across rat trachea in vitro after exposure to cytoskeleton-active drugs in vitro or to ozone in vivo.

Full-length tracheas from Sprague-Dawley rats were exposed to cytoskeleton-active drugs in short-term organ culture, and the permeability of the tracheal epithelium was measured by instilling radiotracers into the lumen and assay of the radioactivity appearing in the external bathing medium. In vitro treatment with cytochalasin D (cyto D, 2-10 x 10(-6) M) increased the rate of movement of [14C]mannitol across the epithelium. Exposure to vinblastine (VB, 10(-4) M) alone had no significant effect. However, VB in combination with cyto D increased the permeability in a dose-dependent manner. In vivo exposure to ozone (O3, 0.8 or 2.0 ppm, 2 h) had only a slight effect on the rate of movement of the tracer as measured in vitro immediately after exposure. At 24 h postexposure there was no significant difference in permeability between ozone- and air-exposed tracheas. Prior in vivo O3 exposure sensitized the tracheas to the in vitro effects of cyto D; treatment of O3-exposed tracheas with cyto D immediately after O3 exposure produced a greater than additive effect on permeability measured in vitro. VB at concentrations up to 10(-4) M had no enhancing effect on permeability in O3-exposed tracheas. Sham exposure to clean air did not affect permeability compared to untreated (shelf) controls. Electron microscopic studies demonstrated penetration of horseradish peroxidase into intercellular spaces in the tracheas treated in vitro with cyto D or cyto D plus VB. Cyto D is known to affect intracellular microfilaments that have attachments at or near the cell surface, while VB affects microtubules associated with internal cellular structures. Therefore, the synergistic effect on tracheal permeability observed with O3 and cyto D, but not with O3 and VB, suggests that O3 may change cell surface structures associated with the microfilamentous cytoskeleton.

Animals↗