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An eight-year study of the viral agents of acute gastroenteritis in humans: ultrastructural observations and seasonal distribution with a major emphasis on coronavirus-like particles.

During an 8-yr period, 862 stool specimens from patients with gastroenteritis were examined by electron microscopy after negative staining with 2% phosphotungstic acid (pH 6.5). Forty-one percent of the specimens submitted over an 8-yr period were determined to be positive for virus or viruslike particles belonging to one or more of seven morphologically distinct viral groups. Coronavirus-like particles (CVLPs) were present in 69.8% of the positive stool specimens. Membranous profiles containing "complement-type" holes (10 nm in diameter) were identified in some preparations containing CVLPs. The second most prevalent viral agent found in stool specimens was the rotavirus (17% of all positive stools). The incidence of other viruses identified in the survey were as follows: adenovirus 4.5%, picorna/parvovirus agents 2.9%, Norwalk-like agent 2.9%, astrovirus 1.9%, and calicivirus 0.5%. Unclassified small round viruses (approximately 25-30 nm in diameter) represented 0.5%. It was also determined that there was a seasonal distribution in excretion of all viruses except for CVLPs. A greater number of viruses were identified in the cooler, drier months of the year.

Adenoviruses, Human↗

Development of axonal-oligodendroglial relationships and junctions during myelination of the optic nerve.

The early stages of myelination were examined in optic nerves of rats aged 12-15 days. The initial association between oligodendroglial processes and bare axons involves no junctional specialization, as the axoglial extracellular space remains unaltered. Following ensheathment by a collar of glial cytoplasm, at least one full rotation of mesaxon was evident before compact myelin formed. Furthermore, myelin was generally evident before a second rotation was completed. In longitudinal sections, an axoglial junction was always observed beginning on the first paranodal loop, continuing through to the last (or outermost) loop. Thus, the formation of myelin and elaboration of a junctional complex in the paranodal region follow a promyelination phase and appear to be synchronous (and possibly related) events. Although the paranodal plasmalemma and axolemma are in close apposition, there is a material in the extracellular space that precipitates phosphotungstic acid, a characteristic that appears to be featured in a number of different types of cell junctions.

Animals↗

Ultrastructure of the flagellar basal body complex of Centipeda periodontii.

The morphology and insertion of the flagellar basal body complex into the cell wall of Centipeda periodontii was studied by electron microscopy of both negatively and positively stained specimens. Freshly harvested cells were examined either after treatment with 0.2% sodium dodecyl sulfate (SDS) for 2 min and negatively stained with phosphotungstic acid, or after treatment according to standard electron microscopy procedures that included positive staining. Small numbers of flagella were dislocated from the cell body after treatment with SDS. The flagella demonstrated an unusual basal body structure: five rings were attached to a rod in a three-ringed (distal) and two-ringed (proximal) patterns; ring diameters produced a distinctive hourglass shape. The cell envelope was typical for gram-negative bacteria with a cytoplasmic membrane and an outer membrane separated by a peptidoglycan layer. Basal body length and cell wall width were in general agreement, approximately 29 nm. Cell wall width exceeded dimensions previously reported for Escherichia coli; this was attributed to an unusually thick peptidoglycan layer.

Flagella↗

Leiomyoma of the oral cavity: a light microscopic and immunohistochemical study with review of the literature from 1884 to 1992.

Leiomyoma is the most common benign neoplasm in the uterus and stomach but is rare in the oral cavity. There were only 5 oral cases in a series of 7748 leiomyomas of all sites. Benign smooth muscle neoplasms are classified into leiomyoma (solid leiomyoma), angiomyoma (vascular leiomyoma) and epithelioid leiomyoma (leiomyoblastoma). 6 cases diagnosed as leiomyoma were retrieved from the files of two oral biopsy services over the past 25 years. A light microscopic study including trichrome and phosphotungstic acid haematoxylin (PTAH) stains, and an immunohistochemical study with the following markers: desmin, muscle specific actin, myoglobin, vimentin, S-100 protein, neuron-specific enolase, factor VIII and Ulex europeus were done with suitable controls. The haematoxylin and eosin and Masson's trichrome stains supported a diagnosis of leiomyoma in all 6 cases but PTAH was positive in only 3 of them. The immunohistochemical study confirmed the diagnosis of leiomyoma in 3 cases. The other 3 were identified as granular cell tumour, myofibroma and neurofibroma, respectively. The review of the literature contributed the following data: mean age was 41 and median age 39 in 134/142 patients. A male sex prevalence 72/137 patients (54.0%) was noted. The lips were the most common site with 39 cases (27.46%) followed by the tongue 26 (18.30%), cheeks and palate 22 (15.49%), gingiva 12 (8.45%), and mandible 8 (5.63%). Prognosis of oral leiomyomas is excellent. Immunohistochemistry is a precise and reliable method for definitive diagnosis of oral leiomyoma.

Adolescent↗

Synthesis and anti-microbial activity of pyrazolylbisindoles--promising anti-fungal compounds.

A series of pyrazolylbisindole derivatives have been synthesized by reacting substituted pyrazole aldehydes with substituted indoles using phosphotungstic acid, a Keggin type heteropoly acid as catalyst. The synthesized pyrazolylbisindoles were evaluated for anti-microbial activities. The effect of pyrazolylbisindoles on the mycelial growth of plant pathogenic fungi is revealed. Entries 3c and 3d emerged as the most interesting compounds in this series exhibiting excellent anti-fungal activity.

Anti-Bacterial Agents↗

A cause of falsely low HDL concentrations in HIV-infected patients: increased polyclonal serum immunoglobulin.

OBJECTIVE: To describe the interference of polyclonal hypergammaglobulinemia in a direct HDL-c method. METHODS: HDL-c was measured by a direct method (Roche Diagnostics) in four HIV-infected patients showing polyclonal hypergammaglobulinemia and also by precipitation with phosphotungstic acid and ultracentrifugation at a density of 1.063 kg/L. In addition, an electrophoretic lipid profile was performed by an automated electrophoretic system. RESULTS: Direct HDL-c concentration was very low or even nondetectable in all hyperglobulinemic samples. Ultracentrifugation showed a higher HDL-c concentration, and the electrophoretic lipid profile showed a band corresponding to HDL particles whose cholesterol concentration was in concordance with that of ultracentrifugation. Kinetic monitoring of the absorbance for the direct HDL-c method showed a high reading after the first reagent addition and a final absorbance quite close to or even lower than the initial absorbance. CONCLUSION: Although interference by any HIV therapy drug cannot be completely ruled out, our results showed that when high immunoglobulin concentrations existed in the patient's serum, the direct HDL-c method yielded low or undetectable HDL-c values. The high initial absorbance observed suggested an interaction between immunoglobulins and the first reagent of the method, producing a complex that scattered light and falsely decreased HDL-c concentrations.

Antiretroviral Therapy, Highly Active↗

Subcutaneous microchip-associated tumours in B6C3F1 mice: a retrospective study to attempt to determine their histogenesis.

Fifty-two subcutaneous tumours associated with microchip were collected from three carcinogenicity B6C3F1 mice studies. Two of these 52 tumours were adenocarcinoma of the mammary gland located on the dorsal region forming around the chip. All the other 50 were mesenchymal in origin and were difficult to classify on morphological grounds with haematoxylin-eosin. These sarcomas were investigated using a panel of immunohistochemistry and special stains consisting of desmin, smooth muscle actin (SMA), myogenin, S100, mouse macrophages, phosphotungstic acid haematoxylin (PTAH) and Masson's trichrome (MT). All the sarcomas displayed the same histochemical characteristics and a close immunophenotype, characterized by desmin +/-, SMA+, myogenin--, S100--, mouse macrophages + and PTAH-. These tumours thus appear to have similar histologic-type lineage and designation as sarcomas not otherwise specified (NOS) with a large myofibroblastic component appears today to be more appropriate and it is likely to clarify them in the future with the emergence of new markers.

Animal Identification Systems↗

Conductimetric determination of phenylpropanolamine HCl, ranitidine HCl, hyoscyamine HBr and betaine HCl in their pure state and pharmaceutical preparations.

Sodium tetraphenylborate and phosphotungstic acid were used as titrants for the conductimetric determination of phenylpropanolamine HCl (PPA.Cl), ranitidine HCl (Ra.Cl), hyoscyamine HBr (Hy.Br) and betaine HCl (Be.Cl) through ion-associate complex formation. The molar combining ratio and the solubility products of the formed ion-associates were studied and calculated. The suggested method has been applied to the determination of the mentioned drugs in their pure state and pharmaceutical preparations with mean recovery values of 97.71-102.97% and relative standard deviations 0.25-0.85%. The accuracy of the method is indicated by excellent recovery and low standard deviation. The results are compared with the pharmacopoeial or the official methods.

Atropine↗

Natural killer-like T-cell lymphoma in a calf.

A case of natural killer (NK)-like T-cell lymphoma in a 9-month-old female Holstein calf is described. The liver, spleen and lymph nodes were affected with lymphoma. The neoplastic cells showed not only epitheliotropism in the biliary epithelium and hepatic cords but also preferential homing to follicular centres of the lymph nodes. In the cytoplasm, there were eosinophilic granules of various sizes, which were positive with phosphotungstic acid haematoxylin and naphthol AS-D chloroacetate esterase. Erythrophagia by lymphoma cells was rarely detected. Immunohistochemically, the neoplastic cells expressed surface CD3, surface CD5 and CD57, and perforin expression was present in the cytoplasmic granules. The lymphoma described resembled feline NK-like T-cell lymphoma in epitheliotropism in the liver and phagocytic activity but differed in respect of follicular involvement and marked variation in granule size.

Animals↗

Extended period of asymptomatic prion disease after low dose inoculation: assessment of detection methods and implications for infection control.

We used quantal dose-titration of a mouse-adapted human transmissible spongiform encephalopathy strain (M470) to compare different analytical methods for their ability to detect asymptomatic brain prion infection after low dose inoculation. At a time point approximately 2.5-fold beyond the mean incubation period of high dose inocula, asymptomatic brain infection was commonly observed using histologic examination, Western blot, and "blind" bioassay following intracerebral inoculation with low titer inocula. At this time point, when a clinical end-point titration would usually be determined, evidence of infection was seen in all healthy animals inoculated with up to 100-fold lower inoculation doses than the lowest causing consistent clinical disease. For the assessment of the presence of asymptomatic infection, we compared different Western immunoblot and histopathological methods in relation to "blind" bioassay using transgenic Tga/20 mice overexpressing mouse prion protein (PrP). Sodium phosphotungstic acid (NaPTA) precipitation of protease-resistant PrP isoforms (PrP(res)) prior to Western blotting was found to approach the sensitivity of the Tga/20 bioassay and was superior to conventional Western blot and histopathological methods, wherein infectivity was commonly found when both of the latter were negative. Re-scaling the original titer by incorporating "blind" transmission data from surviving asymptomatic mice revises the estimate two orders of magnitude higher than the value derived using the conventional clinical disease outcome approach. We also found that the sensitivity of the NaPTA Western blot technique, if used with a diluent such as PBS compared with 10% normal brain homogenate, is adversely affected by up to around 20-fold. We postulate that infectious titer estimates based on more sensitive detection systems such as we report provide a more accurate indication of ultimate transmission risk.

Animals↗

Experimental confirmation of calculated phases and electron density profile for wet native collagen.

An experimental procedure is developed to phase the reflections obtained in x-ray diffraction investigations of collagen in native wet tendons. Phosphotungstic acid was used for isomorphous addition in phase determination and was located by electron microscopy. Structure factors (with phases) were obtained from the electron microscopy data for the heavy metal. Structure-factor magnitudes for collagen with and without the heavy metal were obtained from the x-ray diffraction data. The first 10 orders were investigated. Standard Argand diagrams provided two solutions for each of these, except the weak sixth order. In each case, one of the two possible solutions agrees well with the phases proposed on theoretical grounds by Hulmes et al. The present results suggest that their other proposed phases are probably correct. An electron density profile along the unit cell of the fibril is presented that shows a distinct step, as expected on the basis of the hole-overlap model. The overlap region is 48% of the length of the unit cell.

Animals↗

A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma.

The purpose of the present study was to re-evaluate the three-dimensional organization of collagen fibrils and proteoglycans (PGs) in the human corneal stroma using an improved ultrastructural approach. After a short aldehyde prefixation, one half of seven fresh corneal buttons was stained for PGs with Quinolinic Phtalocyanin (QP) or Cupromeronic Blue (CB). Strips of 1 mm width were cut, subsequently treated with aqueous phosphotungstic acid (PTA) and further processed for light and electron microscopy. The other half of the corneas served as control and was routinely processed with OsO4. Embedding was as such that ultrathin sections could be cut precisely parallel (frontal sections) or perpendicular (cross sections) to the corneal surface. The mutual connections between collagen fibrils and PGs were studied and the length of PGs and their mutual distance were measured manually at a calibrated final magnification of 70,000 x. Prefixed fresh corneal tissue treated with QP and CB shows no signs of swelling and exhibits well contrasted PGs. In cross sections PGs form a repeating network of ring-like structures (approximately 45 nm) around the collagen fibrils. In frontal sections PGs are aligned orthogonal to the collagen fibrils, are equidistant (approximately 42 nm) attached to the collagen fibrils along their full length and have a thickness of approximately 11 nm and a length of approximately 54 nm. The observed maximal length of the PGs and the occurrence of ring-like structures enwrapping the collagen fibrils urged us to revisit the prevailing model of maurice (1962) on the organization of the corneal stroma. In the new model hexagonal arranged collagen fibrils are interconnected at regular distances with their next-nearest neighbours by groups of six PGs, attached orthogonal to the circumference of the fibrils. In this way a regular meshwork of ring-like structures enwrapping the collagen fibrils is formed. It is discussed that this new model more convincingly explains corneal resistance to compression and stretching and further rationalizes corneal transparency because of the low refractive index difference between the regularly arranged collagen fibrils and their inter-space filled with PGs.

Aged↗

Eosinophilic neuronal inclusions in the thalamus of ageing B6C3F1 mice.

The age-related incidence of thalamic neuronal inclusions in the brains of aged B6C3F1 mice and their histopathological and ultrastructural features were studied. Round to oval or rod-shaped eosinophilic inclusions, which were frequently observed in the neurons of the thalamic area, were stained positively with phosphotungstic acid haematoxylin. The inclusions were detected first at 32 weeks of age and the incidence and severity were higher in older animals, all of the mice being affected after 58 weeks of age. Ultrastructurally, the inclusions appeared as sheaves of parallel osmiophilic filaments in the perikaryon of the neurons in the thalamus. Thus thalamic neuronal inclusions in mice seem to be age-related, but their significance remains unclear.

Aging↗

Molecular conformation and packing in collagen fibrils.

New X-ray diffraction data have been collected from specimens of tendon collagen stained with phosphotungstic acid. Measurements of the positions of the Bragg reflections associated with the crystalline lattice provide, for the first time, a complete description of the unit cell. A strong band of intensity in the molecular transform associated with the pitch of the molecular helix can be identified and a detailed analysis of the intensities and positions of the Bragg reflections in this band has been carried out. The principal conclusions are that the portions of the collagen molecule that contribute to these reflections have a common direction; that they have a length very much less than that of a complete molecule; that the paths of the individual portions through the crystal are incompatible with a completely straight molecule, and that the molecule is therefore crimped. No evidence was obtained for a second series of Bragg reflections attributable to a second set of molecular portions linking the first set, and it is concluded that the linking set is more mobile and subject to positional variation from cell to cell. The most plausible explanation of our finding is that the first set corresponds to the portions of the molecules in the overlap zone and the second set to the portions in the gap zone. A detailed analysis of the Bragg reflections in the strong band of intensity associated with the pitch of the molecular helix has provided information about the relative azimuthal orientations and the lateral positions in the unit cell of the five molecular segments in the overlap zone. None of the existing models for fibril structure accounts satisfactorily for all the results obtained in the present studies and alternative models are developed and tested.

Animals↗

Transplacentally induced anorectal malformations in rats.

Twenty-eight pregnant rats (Wistar-Imamichi) on the 11th gestation day were treated with a single intragastric administration of ethylenethiourea (ETU) of 100, 125, 150, and 200 mg/kg body weight, and received cesarean section on the 20th gestation day. No dam died following the ETU treatment. In these transplacentally treated rat fetuses, various types of externally visible malformations were frequently observed; absent or kinked short tail in 100% and spina bifida or myeloschisis in 48.6% to 86.8%. The incidences of these anomalies were significantly higher in males than females. The fetuses were fixed in Bouin's solution and embedded in paraffin, and the step-serial section were sagittally prepared. Then they were stained with phosphotungstic acid hematoxylin for the light microscopic examination. In a preparation of just midsagittal section, malformations of rectum and anus were investigated. The results were as follows: (1) the most appropriate dose of ETU might be 125 mg/kg, body weight of dam, which was suggested by number of the fetuses, prenatal development of the fetuses and incidences of externally visible anomalies in the fetuses; (2) various types of anorectal anomalies, including rectourethral fistula, rectocloacal fistula, covered anus-complete, anoperineal fistula, and anal membrane stenosis, were induced; (3) the incidence of anorectal anomalies was significantly higher in males than females; (4) in every type of anorectal anomalies, inner circular layer of smooth muscle was hypertrophied at the rectum end, which seemed to be considered as the internal sphincter muscle; and (5) the external sphincter muscle complex was hypoplastic in the high deformity fetuses associated with sacrococcygeal anomaly.

Anal Canal↗

Ultrastructural and cytochemical study on the enzyme gland of the foot of a mollusc.

The enzyme gland of the foot of the mussel Mytilus has been so far considered a gland producing and exporting a phenol oxidase catalysing the general tanning processes of byssus threads. In contrast, the present study shows that this gland produces mainly secretory granules which form the cortical layers of byssus threads. Cytochemical methods at the ultrastructural level (phosphotungstic acid at low pH, silver methenamine, periodic acid-thiosemicarbazide-silver proteinate, silver methenamine for sulphur-rich proteins demonstration) and enzyme digestion tests (pepsin, trypsin, alpha-chymotrypsin) indicate that secretory granules contain glycoproteins rich in sulphydryl groups and in aromatic amino acids. The cytochemical demonstration of phenol oxidase shows that enzyme activity is present in Golgi complex, whereas it is absent in secretory granules. For this reason, phenol oxidase does not seem to be exported and utilized for tanning of byssus threads, but it might rather be involved in the elaboration and tanning of the content of the secretory granules in the enzyme gland itself.

Amino Acids↗