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Morphological and phenotypical characterization of Bacillus sporothermodurans.

AIMS: Enumeration of resistant bacteria in ultra-high temperature (UHT) treated milk; morphological characterization and phenotyping of resistant strains by traditional and nontraditional methods and their identification by molecular biology. METHODS AND RESULTS: Modified standard plate count agar (PCA) and modified brain-heart infusion (BHI) agar were used for colony counts. Physiological culture traits were determined as suggested by Bergey's Manual of Systematic Bacteriology or in modified J-broth or in modified BHI agar. Scanning electron microscope (SEM) was used for microscopic examination. Strain identification was carried out by polymerase chain reaction (PCR). A total of 125 (62.81% of 199) samples were positive and the bacterial load was higher than 10(5) CFU ml(-1) in 46 samples (28.80% of 125). The 16S rRNA sequence of bacterial cultures obtained from UHT-treated milk was similar to that of Bacillus sporothermodurans M215 type strain((T)) and different biotypes were found by analysis of colony appearance, cell morphology and physiological traits. CONCLUSIONS: Bacillus sporothermodurans was the predominant sporigenous micro-organisms in UHT milk. SIGNIFICANCE AND IMPACT OF THE STUDY: BHI agar is more suitable than PCA for quality control of milk after UHT treatment. Modified J-broth medium is useful to determine selected physiological traits of B. sporothermodurans. The strains characterized and identified as B. sporothermodurans were significantly different compared with the type strain.

Animals↗

Intraoperative laparoscopic sonography for improved preoperative sonographic pathologic characterization of adnexal masses.

This study compares the diagnostic accuracy of laparoscopic sonography and transvaginal sonography in the evaluation of adnexal masses. Fifty-eight women underwent transvaginal sonography, which showed 69 adnexal masses, and laparoscopic ultrasonography, which showed 68 adnexal lesions. Conventional gray-scale ultrasonography (using transvaginal sonography and laparoscopic ultrasonography) was performed with morphologic characterization of internal architecture, followed by color Doppler imaging with spectral Doppler analysis where possible. A specific diagnosis was obtained with transvaginal sonography and laparoscopic ultrasonography based on a combination of imaging features. The specific diagnosis obtained with each imaging modality was compared with the final histologic diagnosis as the gold standard in 57 patients with 68 adnexal masses who underwent cystectomy or oophorectomy. The ability of laparoscopic sonography to detect the contralateral ovary and any residual ovarian tissue in the presence of a mass was also compared with transvaginal sonography. The accuracy of laparoscopic ultrasonography in the characterization of adnexal masses was 83.8% and that of transvaginal sonography was 73.5% (P < 0.05). Laparoscopic sonography showed greater morphologic detail than that obtained with transvaginal sonography, allowed more precise and specific characterization of adnexal masses, and detected additional adnexal lesions not evident on preoperative transvaginal sonography. Laparoscopic ultrasonography showed the contralateral ovary in 86.2% of patients, compared with 81.0% using transvaginal sonography (P = 0.51). In addition, laparoscopic ultrasonography was able to demonstrate the presence of residual ovarian tissue in the side affected pathologically in 76.5% of patients compared with 59.4% using transvaginal sonography (P < 0.005). Laparoscopic sonography allows more precise morphologic characterization of internal architecture and histologic diagnosis of adnexal lesions, but it is as yet unable to increase the diagnostic accuracy of borderline or malignant lesions, possibly due to the small sample size. Laparoscopic sonography is superior to transvaginal sonography in the evaluation of residual ovarian tissue in the side affected pathologically, which may help in surgical planning between cystectomy and oophorectomy, and also in the identification of the contralateral ovary, which may potentially increase the detection of bilateral pathologic conditions.

Adnexal Diseases↗

Distribution and processing of the polymeric immunoglobulin receptor in the rat hepatocyte: morphological and biochemical characterization of subcellular fractions.

The transepithelial transport of polymeric immunoglobulins is an essential process in the mucosal immune system. Transport across the epithelial cells of mucous or exocrine glands is affected by an integral membrane glycoprotein receptor known as membrane secretory component (SCm) or as polymeric immunoglobulin receptor (pIgR). This receptor binds polymeric immunoglobulins at the basolateral cell surface and mediates their transcellular translocation and their release from the apical plasma membrane into external secretions. Release depends on cleavage of the membrane-anchoring domain of the receptor, resulting in liberation of polymeric immunoglobulin bound to the ectoplasmic domain of the receptor (secreted SC or SCs) into extracellular secretions. Using a monoclonal antibody directed against the cytoplasmic tail of the receptor and a polyclonal antibody directed against the secreted ectoplasmic domain, we have combined cell fractionation and Western blotting techniques to examine the fate of these receptor domains in the hepatocyte. In this study, we characterize biochemically and morphologically the various subcellular components separated by our fractionation scheme, and correlate this with biochemical analysis of the receptor in each fraction.

Animals↗

Lucifer yellow filling of immunohistochemically pre-labeled neurons: a new method to characterize neuronal subpopulations.

We describe a new technique for the morphological characterization of immunohistochemically labeled neuron populations. We demonstrate that it is possible to fill neurons iontophoretically with Lucifer Yellow (LY) in fixed slices of cat visual cortex after the respective cells have been identified by indirect immunofluorescence for the neural cell adhesion molecule N-CAM 180, with the VC1.1 antibody or with an antibody against glutamate dehydrogenase (GAD). Morphological analysis of the injected cells at the light and electron microscopic level revealed that the N-CAM 180-positive neurons share the features of neuropeptidergic cortical interneurons. Depending on the antibody applied, the immunohistochemical treatment had little or no noticeable effect on the quality of LY filling or on the preservation of morphological details of the pre-labeled cells. This makes the method described ideally suited for the light and electron microscopic examination of selected, immunologically characterized neuron subpopulations.

Animals↗

Age-related degeneration of the serotoninergic fibers in the zitter rat brain.

The serotonin neuron system was studied using immunohistochemical and neurochemical techniques in zitter mutant rats aged 1-14 months, which are characterized by abnormal metabolism of superoxides. The morphology of the serotoninergic neuron system and the serotonin level in the zitter rat were compared to those of age-matched Sprague-Dawley (SD) rats. Up to age 4 month, the density and distribution of serotoninergic fibers in the zitter rat brain were similar to those of control rats. However, several serotoninergic fibers with abnormal morphology, characterized by swollen varicosities, were observed in the cerebral cortex and caudate putamen of 6-month-old zitter rats. The density and distribution of these fibers in other regions of the brain were similar to those of control rats. The abnormal serotoninergic fibers increased in number and extended into other regions of the brain such as the thalamus, hippocampus, and vestibular nucleus. On the other hand, the density of normal serotoninergic fibers decreased throughout the brain of the 14-month-old zitter rat. Abnormal serotoninergic fibers have also been reported in the brain of normal older (24 months) SD rats. Neurochemical analysis revealed lower levels of serotonin, and 5-hydroxyindoleacetic acid, in all cortical areas (prefrontal, parietal, and occipital cortices), the caudate putamen, and the hippocampus of 12-month-old zitter rats. Levels differed significantly in the parietal cortex and hippocampus between the zitter and SD rats. Based on the morphological and neurochemical similarities, the present results suggest that age-related degeneration of serotoninergic fibers occurs in the zitter rat brain. Furthermore, degeneration of serotoninergic fibers appears to be induced by superoxide species. Thus, the zitter rat may provide a good model for studying the neurotoxic effects of superoxide species on the serotoninergic neuron system.

Aging↗

Postnatal growth of the ventral prostate in Wistar rats: a stereological and morphometrical study.

Morphological and stereological analyses were used to characterize the growth kinetics of the Wistar rat ventral prostate (VP). Volume density and absolute volume of the epithelium, lumen, smooth muscle cells (SMCs), and nonmuscular stroma were determined by stereology and paired with plasma testosterone levels and different morphometric measurements. The VP shows an initial growth within the first 3 weeks, a resting phase, and the puberal growth. The puberal growth was coincident with the raise in plasma testosterone. Lumen formation occurred within the 3 postnatal weeks. After an expected increase during puberty, the lumen showed a further increase at the 12th week. The volume density of the nonmuscular stroma and of the SMCs decreased slowly postnatally. Absolute volume of the luminal compartment showed three phases of growth (weeks 1-3, 6-9, and 11-12). On the other hand, the increase in the absolute volume of the epithelium was steady up to the 8th week and then showed a marked increase up the 10th week. The increase in epithelial volume was characterized morphologically by the presence of epithelial infoldings and sprouts. The growth of the epithelium showed a 2-week delay as compared to the lumen and occurred only until the 10th week. The epithelial height was variable but could be related to the synthetic activity of the epithelium. In conclusion, the postnatal growth of the VP results from a combination of epithelial proliferation/differentiation and synthesis/accumulation of the secretory products in the lumen.

Animals↗

Polyphosphate content and fine structure of acidocalcisomes of Plasmodium falciparum.

Although acidocalcisomes have been well characterized morphologically in other apicomplexan parasites, no such characterization has been done in Plasmodium spp. Here, we report that Plasmodium falciparum merozoites possess electron-dense organelles rich in phosphorus and calcium, as detected by X-ray microanalysis of intact cells, which are similar to the acidocalcisomes of other apicomplexans, but of more irregular form. In agreement with these results malaria parasites possess large amounts of short- and long-chain polyphosphate (polyP), which are associated with acidocalcisomes in other organisms. PolyP levels were highest in the trophozoite stage of the parasite. Treatment of isolated trophozoites with chloroquine resulted in a significant hydrolysis of polyP. Taken together, these results provide evidence that acidocalcisomes from Plasmodium falciparum do not differ significantly from acidocalcisomes of other apicomplexan parasites.

Animals↗

[Quantitative description of the morphological structure of aggregated cellular elements in blood].

We introduce indices which characterize morphological structure of the system of aggregated cells in a thin blood layer. The main quantitative characteristics of the system is the multifractal spectrum f(alpha). Analysis of the f(alpha) profile for varying aggregation moments allows to describe quantitatively the dynamics of formation and growth of cell aggregates as well as to determine the structural morphological changes during this aggregation. Other quantitative values describing the aggregation kinetics are the scaling exponents characterizing the power dependence of the cluster quantity ns(t) on the number of particles in the cluster s and on the time moment t.

Blood Cells↗

Morphologic, cytochemical and neurochemical characterization of the human medulloblastoma cell line TE671.

Medulloblastoma cell line TE671 was characterized by morphologic, cytochemical, neurochemical, and growth criteria. In contrast to the uniform, in vivo histopathologic appearance of the tumor, TE671 in vitro exhibits six morphologic subtypes (Types I-VI) in varying percentages over 14 days in culture. TE671 grows as a monolayer by the merging of separate foci. Cells were positive for Periodic acid Schiff (PAS) and reticulum, and negative for the glial marker, glial fibrillary acid protein (GFAP). Receptors for human C3b (EAC) were present on 19% of the cells. Neural associated isoenzymes, neuron specific enolase (NSE) and creatine kinase (CK-BB) were demonstrated in TE671. Progeny of a single clonogenic cell manifested the morphologic heterogeneity of cell types (I-VI). The absence of markers specific for glial cells suggests that TE671 is an early (less differentiated) precursor. TE671, the only continuous human medulloblastoma cell line, provides an experimental model with which to compare and identify the subpopulation of neoplastic cells in medulloblastoma ex vivo.

Cell Line↗

Morphological, physiochemical and molecular characterization of Anabaena strains.

A set of 30 Anabaena strains, isolated from diverse geographical regions of India, were characterized using morphological and physiochemical attributes as well as molecular marker profiles. Significant differences were observed among the Anabaena strains with regard to the shape and size of trichomes and individual cells within a filament, besides qualitative and quantitative aspects of phycobiliprotein accumulation and activities of enzymes involved in nitrogen metabolism. Analyses of molecular polymorphisms in a selected set of 13 Anabaena strains, using primers based on repetitive sequences in the genome, led to unambiguous differentiation of the strains as well as understanding of their genetic relationships. Informative morphological, physio-chemical and molecular characters have been identified that could aid in differentiation and utilization of Anabaena strains as bioinoculants or as sources of pigments.

Anabaena↗

Morphological, ontogenetic and molecular characterization of Scutellospora reticulata (Glomeromycota).

The arbuscular mycorrhizal (AM) fungus Scutellospora reticulata (CNPAB11) was characterized using morphological, ontogenetic and molecular approaches. Spore ontogenesis was studied using Ri T-DNA transformed carrot roots and observations were compared with those published for eight other, pot-cultured, Scutellospora species. The sporogenesis of S. reticulata exhibited an unreported pattern of outer spore wall differentiation. In addition, Denaturing Gradient Gel Electrophoresis (DGGE), targeting the V9 region of the SSU nrDNA, was used to differentiate S. reticulata from 16 other Scutellospora species and results were confirmed by sequencing analysis. Phylogenetic analyses, using nearly full length SSU nrDNA sequences, grouped S. reticulata in a cluster together with S. cerradensis and S. heterogama, species that share similar spore wall organization and also possess ornamented external walls. PCR-DGGE and sequence analysis revealed intragenomic SSU nrDNA polymorphisms in four out of six Scutellospora species tested, and demonstrated that SSU nrDNA intragenomic polymorphism could be used as a marker to differentiate several closely related Scutellospora species.

DNA, Fungal↗

Mechano-morphological studies of aligned nanofibrous scaffolds of polycaprolactone fabricated by electrospinning.

Mechanical and morphological studies of aligned nanofibrous meshes of poly(epsilon-caprolactone) (PCL) fabricated by electrospinning at different collector rotation speeds (0, 3000 and 6000 rpm) for application as bone tissue scaffolds are reported. SEM, XRD and DSC analyses were used for the morphological characterization of the nanofibers. Scaffolds have a nanofibrous morphology with fibers (majority) having a diameter in the range of 550-350 nm (depending on fiber uptake rates) and an interconnected pore structure. With the increase of collector rotation speed, the nanofibers become more aligned and oriented perpendicular to the axis of rotation. Deposition of fibers at higher fiber collection speeds has a profound effect on the morphology and mechanical properties of individual fibers and also the bulk fibrous meshes. Nanoindentation was used for the measurement of nanoscopic mechanical properties of individual fibers of the scaffolds. The hardness and Young's modulus of aligned fibers measured by nanoindentation decreased with collector rotation speeds. This reveals the difference in the local microscopic structure of the fibers deposited at higher speeds. The sequence of nanoscopic mechanical properties (hardness and modulus) of three fibers is PCL at 0 rpm > PCL at 3000 rpm > PCL at 6000 rpm. This may be explained due to the decrease in crystallinity of fibers at higher uptake rates. However, uni-axial tensile properties of (bulk) scaffolds (tensile strength and modulus) increased with increasing collector rotation speed. The average ultimate tensile strength of scaffolds (along the fiber alignment) increased from 2.21 +/- 0.23 MPa for PCL at uptake rate of zero rpm, to a value of 4.21 +/- 0.35 MPa for PCL at uptake rate of 3000 rpm and finally to 9.58 +/- 0.71 MPa for PCL at 6000 rpm. Similarly, the tensile modulus increased gradually from 6.12 +/- 0.8 MPa for PCL at uptake rate of zero rpm, to 11.93 +/- 1.22 MPa for PCL at uptake rate of 3000 rpm and to 33.20 +/- 1.98 MPa for PCL at 6000 rpm. The sequence of macroscopic mechanical properties (tensile strength and modulus) of three fibers, from highest to lowest, is PCL at 0 rpm < PCL at 3000 rpm < PCL at 6000 rpm. This is attributed to the increased fiber alignment and packing and decrease in inter-fiber pore size at higher uptake rates.

Biocompatible Materials↗

Hepatocyte cell surface polarity as demonstrated by lectin binding.

We performed an investigation at the ultrastructural level of the differential distribution of lectin-binding sites among sinusoidal, lateral, and bile canalicular domains of adult rat hepatocytes. Lectin binding to hepatocyte glycocalices was studied in situ or after cellular dissociation by enzymatic (collagenase), chemical (EDTA), and mechanical methods, as well as during cell culture. Using thirteen biotinylated lectins and an avidin-biotin-peroxidase complex (ABC), we have identified lectin-binding sites that are predominantly localized in the bile canalicular [Ricinus communis agglutinin (RCA)] or sinusoidal [Phaseolus vulgaris (PHA)] domains in situ and in mechanically dissociated cells. Lens culinaris (LCA) staining was prominent on sinusoidal surfaces, slight along lateral surfaces, and completely absent in the bile canalicular domain. Concanavalin A (ConA) was unique in binding equally to all domains. Triticum vulgaris [wheat germ agglutinin (WGA)] was also bound to all domains, but most intensely to the bile canalicular region. Cells dissociated via collagenase or EDTA treatment exhibited a spherical morphology characterized by many surface microvilli and absence of morphological domains. Lectin binding to dissociated cells was uniformly distributed over the entire cell surface, suggesting a redistribution of lectin receptors that was independent of the separation procedure. Hepatocytes in culture exhibited a partial restoration of morphological domains, but lectin binding polarity was not re-established.

Animals↗

[Residual mosquitoes in the northern Adriatic seacoast 50 years after the disappearance of malaria].

The Northern Adriatic Sea littoral was heavily malarious; intensive land drainages, agricultural development and socioeconomic improvement were the key factors which led to malaria eradication, sped up by indoor insecticide spraying, achieved soon after World War II. Regular observations on anophelism were carried out by the Istituto Interprovinciale per la Lotta Antimalarica nelle Venezie from middle 20's until early 60's. The main vector was Anopheles sacharovi, a species which typically bred in coastal brackish swamps; other species were An. atroparvus (which was a probable secondary vector) and the usually strictly zoophilic An. maculipennis, An. melanoon, An. messeae and An. subalpinus. From 1995 to 1997 surveys were carried out in order to review the genus Anopheles in the coastal area of Friuli-Venezia Giulia and Veneto regions. A total of 11,346 females were collected from animal shelters (cow-shed, pigsties, horse stables) of 52 sites along 180 km of coast crossing 5 provinces (from North: Gorizia, Udine, Venezia, Padova and Rovigo). All specimens belonging to the An. maculipennis complex were scored for the presence of the differential characters of An. sacharovi, the only species of the complex morphologically characterized at the adult stage. The examination of morphological characters of single egg batches obtained from field collected females was the main diagnostic tool for the other species. Species identification was subject to confirmation by larval chaetotaxy analysis (number of branches of antepalmate hairs of IV and V abdominal segments) in representative samples of laboratory-reared mature larvae, while biochemical analysis (enzyme electrophoresis) on some samples of identified females was performed in the laboratory of Prof. L. Bullini and Dr. R. Cianchi of the University of Rome "La Sapienza" and partly in our laboratory. No An. sacharovi female was recorded. The examination of 6,361 single ovipositions led to the identification of three species of the An. maculipennis complex: An. atroparvus, An. maculipennis and An. messeae; An. claviger s.str. was also recorded. Larval chaetotaxy examination carried out on 1,608 larvae and the biochemical identification of 467 females confirmed the previous diagnosis based on egg characters. The relative frequency of the three species varied depending on the site: An. maculipennis was the most abundant species north of Venice; south of Venice, and particularly in the Po river delta, the most abundant species were An. atroparvus and, in some sites, An. messeae. In view of the high density recorded for An. atroparvus in some sites (corresponding to various thousands females in a single animal shelter), the vectorial capacity values may be significant and should be assessed.

Animals↗

[Focal segmental glomerular hyalinosis: a special form of fibroplastic glomerulonephritis].

Focal segmentary glomerular hyalinosis (FSGH) presenting a picture of glomerulonephritis of the nephrotic type is characterized morphologically. Clinico-morphological criteria for diagnosis are presented. FSGH is characterized by a severe nephrotic syndrome, progressive course, and refractoriness to immunosuppressive therapy, focal and segmentary pattern of changes in the glomeruli, the presence of hyalin masses in some lobules of glomerular capillaries, lipid inclusions in mesangial and endothelial cells, tubular epithelial cells and interstitium, as well as uneven outlines of the endothelial surface of the basal membrane of glomerular capillaries. The diagnosis of early changes in FSGH was shown to require light and electron microscopic and immunohistochemical examinations of kidney biopsies. The problems of the etiology and pathogenesis of FSGH and its independence as a nosological entity are discussed.

Adolescent↗

Nonlymphoid intraepidermal mononuclear cell collections (pseudo-Pautrier abscesses): a morphologic and immunophenotypical characterization.

We evaluated the incidence, morphology, and immunophenotype of intraepidermal collections of mononuclear cells (ICMC) in a large number of inflammatory dermatosis and cutaneous lymphomas. ICMC appeared as small to large aggregates of cells, showing a morphology variable from monocytes to obvious dendritic cells, admixed with rare lymphocytes. ICMC were recognized in the epidermis or within hair follicle epithelium, and were either loosely or compactly arranged. ICMC were identified in 124 of 1,248 skin biopsies (9.9%) of inflammatory or lymphoid infiltrates, and were particularly frequent in spongiotic (43.4%) and in lichenoid dermatitis (10%), whereas they were rarely found in nonspecific superficial dermatitis (3.8%) and in psoriasis (4.7%). ICMC were also frequent in cutaneous T-cell lymphoma (13.3%), where they mimicked Pautrier abscesses. The ICMC forming cells showed a unique phenotype: the majority of them expressed CD1a and S-100, and lacked CD14, similar to mature Langerhans cells, but they were also strongly labeled by anti-CD11b, anti-CD36, and anti-CD68. Moreover, a subpopulation of them expressed CD83, an antigen that is usually absent on Langerhans cells. The occurrence of ICMC is a rather frequent, although hitherto poorly studied, phenomenon, occurring in several dermatosis, but particularly frequent in spongiosis-associated skin reactions. The cells within ICMC are represented by dendritic cells and dendritic cell precursors, whose phenotype indicates their derivation from circulating monocytes and differentiation into mature Langerhans cells.

Antigens, CD↗

Standards of morphological evaluation and histological grading in experimental acute pancreatitis.

Acute pancreatitis is characterized morphologically by edema, hemorrhages, parenchymal necrosis and fat necrosis. The inflammation is accompanied by infiltration of polymorphonuclear leukocytes. According to the absence or presence of necrosis the disease can be divided into interstitial (or edematous) pancreatitis and hemorrhagic-necrotizing pancreatitis. The severity of disease can be graded in the histological sections either by giving scores to the different types of morphological alterations or by determining the proportion of necrotic tissue of the total lobular parenchyma. The former method is based on subjective assessment of histological slides and is suitable for the evaluation of both edematous and necrotizing pancreatitis. Histometric measurement of necrotic parenchyma can be used only in the necrotizing forms of experimental pancreatitis, e.g. in those induced by intraductal injection of bile, bile salts or digestive enzymes, and in the dietary ethionine-induced pancreatitis. Grading of the tissue damage is essential when the effects of different therapies are evaluated.

Acute Disease↗

Expression of the naturally occurring truncated trkB neurotrophin receptor induces outgrowth of filopodia and processes in neuroblastoma cells.

We have investigated the effects of the truncated trkB receptor isoform T1 (trkB.T1) by transient transfection into mouse N2a neuroblastoma cells. We observed that expression of trkB.T1 leads to a striking change in cell morphology characterized by outgrowth of filopodia and processes. A similar morphological response was also observed in SH-SY5Y human neuroblastoma cells and NIH3T3 fibroblasts transfected with trkB.T1. N2a cells lack endogenous expression of trkB isoforms, but express barely detectable amounts of its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). The morphological change was ligand-independent, since addition of exogenous BDNF or NT-4 or blockade of endogenous trkB ligands did not influence this response. Filopodia and process outgrowth was significantly suppressed when full-length trkB.TK+ was cotransfected together with trkB.T1 and this inhibitory effect was blocked by tyrosine kinase inhibitor K252a. Transfection of trkB.T1 deletion mutants showed that the morphological response is dependent on the extracellular, but not the intracellular domain of the receptor. Our results suggest a novel ligand-independent role for truncated trkB in the regulation of cellular morphology.

3T3 Cells↗