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PubMed1981

Negatively stained punctated cell contacts between luteal cells in rabbit and rat.

Cell contacts from rat and rabbit luteal cells have been spread on phosphotungstic acid solution after tissue fractionation or spreading was accomplished by a direct technique avoiding homogenization. Sectioned material was also examined. It was shown by negative staining that the cell contacts were composed of a multitude of bridges. These bridges were either filamentous (condensed form) or clearly showed a larger (expanded form) central particle. The corresponding image in top view showed respectively small and larger granules without any particular arrangement. Large granular structures were also seen on single plasma membranes. Evidence is presented that the condensed and the expanded form are two aspects of the same junctional structure. It is also suggested that both aspects are related to the tightness of the contact. Up to now this type of cell contract has been called "septate-like". However because of the dotted appearance of these contacts after colloidal lanthanum and after negative staining the denomination "punctated cell contact" may be better adapted.

Animals↗
PubMed1981

Protein nitrogen unit precipitation procedure for allergenic extracts: collaborative study.

Protein nitrogen unit (NU) determination is one of the methods used to test and label the concentration of allergenic extracts. This recently standardized method is applicable to all allergenic extracts. One PNU/mL is equivalent to 1 X 10(-5) mg nitrogen determined to be in the material precipitated from 1 mL allergenic extract by phosphotungstic acid (PTA), a protein precipitant. The nitrogen is quantitated by the Kjeldahl method or another analytical method of equivalent accuracy and precision. A collaborative study of the optimized PNU precipitation method in which 6 samples were analyzed in duplicate by 6 laboratories using the Kjeldahl method for the determination of nitrogen yielded a mean of 0.1358 mg N/mL, a repeatability standard deviation and coefficient of variation of 0.0071 mg N/mL and 5.23%, respectively, and a reproducibility standard deviation and coefficient of variation of 0.0188 mg N/mL and 13.84%, respectively. The method has been adopted official first action.

Allergens↗
PubMed1980

[Morphometric investigation on the changes of the paramembranous structures of the CA 1 hippocampal synapses of trained rats, with special reference to the morphological plasticity of the synapses].

Albino rats aged 13 weeks experienced a single training for bright-dark discrimination in a Y-chamber. 24 hours later after a successful training the paramembranaceous structures of the synapses contrasted by phosphotungstic acid were morphometrically analysed. As a result of this investigation a signifikant reduction of the height and the width of the dense projections was to be stated compared to untrained rats. Likewise the length and the width of the postsynaptic ribbon were reduced in trained rats. But this alteration proved not to be significant.

Animals↗
PubMed1980

Electron microscopy of mucin from sputum in chronic obstructive bronchitis.

Sputum from patients with chronic obstructive bronchitis has been fractionated on Sepharose columns after treatment with urea 6 mol/l at pH 12.5 followed by neutralization. The "mucin" fraction, which contained 70% carbohydrate and 30% protein was studied in the electron microscope after staining with phosphotungstic acid. Positively stained, thread-like, irregular contours of the mucin molecules could be demonstrated with lengths varying from 2,000 to 5,000 nm, corresponding to molecular weights between 2 X 10(6) and 6 X 10(6). This correlates fairly well with estimates from the literature of the size of these molecules.

Bronchitis↗
PubMed1993

Collagen-glutaraldehyde interaction as revealed by the D-banding of negatively stained fibrils and computer-drawn band patterns.

Band patterns exhibited under electron microscope by native collagen fibrils fixed with glutaraldehyde (2.5%-5% GA diluted in 0.1M phosphate buffer, pH 7.4) and negatively stained with phosphotungstic acid (1% PTA diluted in the same buffer) were digitized to form both bandings and microdensitometric traces. Collaterally, computer-drawn band patterns and traces were yielded on the basis of the "quarter stagger" model and primary structure of alpha 1(I) and alpha 2(I) tropocollagen chains and by selecting options related to specific collagen-GA interactions. Comparisons between actual and simulated patterns suggest that lysines and hydroxylysines should react with GA residues in a 1:3 ratio, while GA-reactivity of histidyl and tyrosyl residues seem to be excluded. On the other hand, an improvement of simulations was achieved by also selecting hydroxyprolines (in addition to lysines and hydroxylysines), which seemed to react with GA in a 1:1 ratio. Considering the bifunctionality of GA, it is suggested that during fixation, heteropolymers form, composed of GA-hexamers bonded to couples of lysyl and/or hydroxylysyl residues. The hypothesis is advanced of an additional formation of GA-dimers, each bonded to two hydroxyprolines.

Animals↗
PubMed1995

The glycan keratan sulfate in inner ear crystals.

The otoconial matrix (OM) of chicks (Gallus domesticus) inner ear was analyzed. Histochemically the OM was reacted with phosphotungstic acid (PTA) and immunohistochemically with the monoclonal antibody antikeratan sulfate (antiKS). The OM was digested with the enzyme endo-beta-galactosidase (E beta Galase) or separated by 1D and 2D gel electrophoresis. PTA which reacts with glycoproteins precipitated the OM, suggesting that the OM contains glycoproteins. A central core in each crystal had no PTA staining, suggesting that the core lacked glycoproteins. Anti KS antibody stained the OM with increased density in older embryos as determined by color thresholding. E beta Galase, which cleaves the lactosamine repeating units in KS, decreased the immunostain by 30% in the OM and by 20% in the cartilage. The OM from the utricle, saccule and macula lagena contained similar molecular weight bands. Five dense bands in the OM were less dense in tissue and blood controls, suggesting that such bands are enriched in the OM. Isoelectric focusing of the OM showed a negatively charged high molecular weight smear not present in blood and faint in tissue controls. The high affinity of the OM for the cationic PTA stain, the strong immunohistochemical reaction of the OM with anti KS antibody and high molecular weight negative smear in 2D gels taken together suggest that: a) the OM contains large amounts of glycoproteins and glycans, one of which is keratan sulfate, because its immuno stain with antiKS antibody was decreased by the enzyme E beta Galase, b) the utricle, saccule and macula lagena may have similar composition, and c) the concentration of KS may increase gradually until complete mineralization of the OM is reached.

Animals↗
PubMed1995

Evaluating lyophilized human serum preparations for suitability as proficiency testing materials for high-density lipoprotein cholesterol measurement.

OBJECTIVE: To evaluate the suitability of various commercial preparations for use by the College of American Pathologists as survey materials in assessing high-density lipoprotein cholesterol measurement performance. DESIGN: Lyophilized human serum preparations from six vendors (vendors A through F) were evaluated to determine which material(s) best mimicked the commutability of fresh human serum. Two freshly collected unfrozen pools prepared from donor specimens were analyzed concurrently with the vendor materials to identify sources of variation and possible matrix bias. Each material was evaluated using 5 common precipitation reagents (phosphotungstate-magnesium, phosphotungstic acid, dextran sulfate [50K and 500K], and heparin-manganese). To evaluate how each reagent separates lipoproteins in each material, the lipoprotein separation patterns were profiled using high-pressure liquid chromatography and compared with separation patterns observed for the fresh human serum pools. MAIN OUTCOME MEASURES: Similarities in performance characteristics of vendor material(s) were compared with fresh human serum. RESULTS: Two of the six materials gave separation profiles for the lipoproteins similar to the typical patterns observed for human serum. Material from vendor B showed the best commutability across all of the precipitation reagents and had the best combination of low overall variability (10% for level 1 and 9.4% for level 2) and minimal concentration differences among reagents. CONCLUSIONS: Vendor B was selected by the College of American Pathologists to provide materials for use in assessing performance of lipid and lipoprotein testing in the 1994 Comprehensive Chemistry Surveys. This study demonstrates the great variability that different vendor preparations introduce into the measurement of high-density lipoprotein cholesterol. It also emphasizes the effort required to evaluate the suitability of processed materials for use in proficiency testing.

Analysis of Variance↗
PubMed1993

Glutaraldehyde-induced D-band pattern of type II collagen fibrils as revealed by negative staining.

A study was carried out on the negative staining band patterns (1% phosphotungstic acid (PTA) in phosphate buffer, 0.1 M, pH 7.4) exhibited by type II collagen fibrils after treatment with glutaraldehyde (5% in phosphate buffer 0.1 M, pH 7.4) and a comparison was made with the negative staining patterns of glutaraldehyde-fixed type I collagen fibrils. A characteristic D-band pattern was observed for type II collagen fibrils. The gap/overlap ratio was unusually low, with a 0.40 long gap zone and a 0.60 D overlap zone. This banding displayed eleven major light bands instead of the fifteen bands per period observed in the type I collagen patterns. On comparing the two types of microdensitograms, seven negative peaks (light bands) coincided and among them were the peaks corresponding to the N-terminal and C-terminal telopeptide regions. Although less numerous, the negative peaks of type II fibril traces were broader and more marked than those of type I microdensitograms and this feature accounts for the greater stain exclusion capacity of the type II fibrils. This is consistent with the larger quantity of hydroxylysines in type II collagen and, perhaps, with the more abundant hydroxyprolines.

Animals↗
PubMed1994

Sudden infant death syndrome: increased synaptic density in the central reticular nucleus of the medulla.

The medulla was sampled from nine cases of sudden infant death syndrome (SIDS) and from six age-matched control cases without neurological disease. Morphometric analyses were performed on serial Nissl sections through the left half of each medulla to determine the total volume of the hemimedulla and the numerical density of neurons (Nv, neurons per mm3) in the central reticular nucleus. Blocks of tissue from the right half were prepared for electron microscopy using the ethanolic phosphotungstic acid (EPTA) method to stain synaptic contacts. Stereological analyses were performed to determine the Nv of synapses in the central reticular nucleus. Total volume of the hemimedulla did not differ significantly between SIDS cases and controls. However, in the central reticular nucleus of SIDS cases, the Nv of neurons was significantly less than in controls (30%), while the mean profile area of reticular neurons was significantly greater (39%). Synaptogenesis in control cases was characterized by a gradual decrease in the Nv of synapses from approximately 150 million at 40 postconceptional weeks to 110 million at 84 weeks. In SIDS cases the Nv of synapses was significantly greater (38%). These results demonstrate a disorder of synaptogenesis in the central reticular nucleus of SIDS cases. In view of previous reports of a persistence of dendritic spines on reticular neurons in SIDS, the results are consistent with a failure to eliminate normally extraneous synapses during early development.

Female↗
PubMed1995

Cartilage type II collagen fibrils show distinctive negative-staining band patterns differences between type II and type I unfixed or glutaraldehyde-fixed collagen fibrils.

The cross striation of native and reconstituted collagen fibrils is believed to conform to a unique D-band pattern independently of the genetically distinct types of fibril-forming collagens. This investigation focuses on type II native collagen fibrils, whose negative-staining patterns are shown to differ from the usual banding exhibited by type I collagen fibrils. Negative staining with phosphotungstic acid, pH 7.4, was carried out on a) unfixed and b) glutaraldehyde-fixed collagen fibrils isolated from bovine hyaline cartilages. The band patterns obtained and their microdensitograms were compared to similarly processed type I collagen fibrils isolated from bovine fibrous tissues. Only minor differences were observed in unfixed fibrils. In the intraperiod light zones of type II fibrils, two dark bands (interbands X2-Y4 and Y4-Y2) showed different intensities with respect to their homologous bands in type I fibrils. In contrast, a marked difference was shown by glutaraldehyde-fixed fibrils. In comparison with type I fibrils, the greater stain exclusion capacity of type II fibrils yielded both the appearance of supernumerary bands, which altered banding in two intraperiod regions, and differences in the intensity of several bands in three intraperiod regions where the band distribution was similar. This stain exclusion pattern may be accounted for by molecular extradensity. The possibility that it depends on linkage with a higher number of glutaraldehyde residues and/or the persistence of cross-linked collagenic or non-collagenic proteins is discussed. To refer to the glutaraldehyde-induced band patterns in negatively stained type II and type I collagen fibrils, the terms "bands GA(II) 1-12" and "bands GA(I) 1-15," respectively, are proposed.

Animals↗
PubMed1979

[Electron microscopic representation of the pili structure of Neisseria gonorrhoeae (author's transl)].

The technique of negative staining and ultra-thin section has been used for investigations of 30 Neisseria gonorrhoeae strains in order to represent the structure of pili (fimbriae) electron microscopically. The staining of the gonococci was effected by phosphotungstic acid (0,5%). The pili ascertained were 30 to 60 A thick. In course of in vitro passages up to 10. subculture morphological changes of the pili have been observed. The application of trisbuffer or solution of Hylase (hyaluronidase) showed not any improved results in comparison with buffered NaCl-solution as suspension medium. The investigation of ultra-thin sections showed that the structure of the pili could be exhibited not clearly. Therefore, these technique seems to be not suitable for qualitative representative of the pili.

Neisseria gonorrhoeae↗
PubMed1996

Atomic force microscopic study on topological structures of pBR322 DNA.

Plasmid pBR322 DNA (0.5 mg/mL) isolated from Escherichia coli HB101 was suspended in Tris-HCI-EDTA (1 mol/L-0.1 mol/L, pH 8.5); then a drop of the above solution was deposited on freshly cleaved mica substrate. After adsorption for about 1 mm, the sample was stained with phosphotungstic acid. The residual solution was removed with a piece of filter paper. Afterwards the sample was imaged with a home-made atomic force microscope (AFM) in air. The AFM images of pBR322 DNA with a molecular resolution have been obtained. These images show that pBR322 DNA exists in several different topological structures: (i) relaxed circular DNA with a different diameter; (ii) supercondensed DNA with different particle sizes; (iii) dimeric catenane connected by one relaxed circular molecule and another close-compacted molecule which might be either supercoiled or intramolecular knotted form; (iv) oligomeric catenane with multiple irregular molecules in which DNA is interlocked into a complex oligomer; (v) possibly-existing intermediates formed from monomer to oligomer.

DNA, Bacterial↗
PubMed1996

Cytochemical and immunocytochemical characterization of kinetochores in the holocentric chromosomes of Graphosoma italicum.

In the present paper we have analyzed the trilaminar kinetochores present in the mitotic holocentric chromosomes of the heteropteran Graphosoma italicum by means of different cytochemical and immunocytochemical techniques at the ultrastructural level. The results obtained after EDTA and osmium tetroxide/p-phenylenediamine, both cytochemical methods for preferential detection of ribonucleoproteins, show that the inner and outer kinetochore layers are selectively contrasted. These results agree with those previously reported for the kinetochores of monocentric chromosomes. Additionally, using cytochemical techniques such as ethanolic phosphotungstic acid staining and silver impregnation, which recognize proteins, we report a different response of the inner kinetochore layer compared with those obtained in the middle and outer layers. Finally, our immunoelectron microscopic results employing monoclonal antibodies against DNA indicate the absence of this component from the kinetochore plates in holocentric chromosomes. Although the trilaminar appearance of kinetochores in holocentric chromosomes is very similar to that of the localized kinetochores in mammals, our observations imply the existence of significant differences on the macromolecular organization of both types of kinetochores.

Animals↗
PubMed1996

[Perforated synapses of the neocortex and their role in the reorganization of the interneuronal relations in the postischemic period (a morphometric study)].

The influence of sudden systemic hemostasis on the change dynamics of numerical density of perforated (PS) and non-perforated (NS) interneuronal synaptic contacts in cerebral sensomotor cortex layer 1 were studied in albino rats during 30 postischemic days using electron microscopy and morphometric methods of examining the material treated with osmium and contrasted with phosphotungstic acid. Regularities of junction and disjunction mechanisms realization in arising PS from NS in diverse postischemic terms were demonstrated. A significant role of PS appearance in reorganization of neocortical neuronal interrelations is shown.

Animals↗
PubMed1996

[Study on the synaptonemal complexes in spermatocytes of the brown rat (Rattus norvegicus caraco). II. Study on the behavior and morphology of the sex chromosomes pairing].

The behavior and morphology of sex chromosomes pairing in spermatocytes of the brown rat have been studied by surface spreading, silver and phosphotungstic acid(PTA) staining techniques. The results are as follows. Sex chromosomes axial cores thicken before X and Y pairing. X Y pairing is delayed until early pachytene. The first pairing initiation site is located on telomere of the short arms of X and Y chromosomes. The second pairing initiation site is located on teltomere of the long arms of X and Y chromosomes or on the interstitial position of the long arms of X and Y. Pycnosis speed of sex axial cores is different with different stages during prophase I of meiosis. In middle Pachytene the almost whole Y is fully paired in SC association with about one-third the X. On the region of X-YSC, lateral elements of X-YSC divide into two skeins, bubble appears on one of the two skeins and the unpaired X and Y axial cores are in variable forms. X Y pairing initiation sites, relationship of X Y pairing and genetic homology and mechanism of thickening and variable X Y axial cores were discussed.

Animals↗
PubMed1997

A new and improved "quick-hot Gram-chromotrope" technique that differentially stains microsporidian spores in clinical samples, including paraffin-embedded tissue sections.

OBJECTIVE: This report describes a new and improved "quick-hot Gram-chromotrope" staining technique that detects microsporidian spores in clinical specimens, such as stool, urine, saliva, nasopharyngeal fluid, and bronchoalveolar lavage samples, as well as in formalin-fixed and paraffin-embedded tissue sections. DESIGN: In this procedure, the samples are stained in heated (50 degrees C to 55 degrees C) solutions of crystal violet and iodine used in Gram's stain, followed by a modified chromotrope solution (heated to 50 degrees C to 55 degrees C). The modified stain is composed of chromotrope 2R (1%), fast green (0.15%), and phosphotungstic acid (0.25%). RESULTS: With this stain and the new protocol, microsporidian spores are stained dark violet against a pale green background, and the total staining time is shortened to 5 minutes. CONCLUSIONS: This new technique is fast, reliable, and simple. It can be easily adapted for use in clinical laboratories.

Animals↗
PubMed1997

A comparative study on morphology and immunohistochemistry of rhabdomyosarcoma and embryonal skeletal muscles.

OBJECTIVE: To investigate the histologic and histogenetic relationship between various types of rhabdomyosarcomas (RMS) and embryonal skeletal muscle (ESM) for further understanding of the histogenesis, classification and pattern of differentiation of RMS. METHODS: Fifty cases of variant types of RMS and 20 cases of ESM at different gestational ages were available. All specimens were stained with HE, PAS, Van Gieson, Masson, phosphotungstic acid hematoxilin and with antibodies for the demonstration of vimentin, desmin, HHF-35 and myoglobin by ABC method. RESULTS: The results showed that the order of positive expression and the intensity of positive reaction of the different immunohistochemical staining were consistent with the degree of differentiation of the tumor and the development of the ESM. It is obvious that each type of RMS is composed of tumor cells in different degree of differentiation and is derived from primitive mesodermal cells which are capable of potential differentiation towards mature skeletal muscles. CONCLUSIONS: Based on the results of this study, an ideal histologic classification of RMS should reflect not only the cell morphology and histologic structures but also the degree of differentiation of the tumor cells.

Adolescent↗
PubMed1998

In situ anatomical study of the male urethral sphincteric complex: relevance to continence preservation following major pelvic surgery.

PURPOSE: We describe a correlative gross anatomical and histological study of the human male urethral sphincteric complex using methods that delineate skeletal, muscular and fascial components. MATERIALS AND METHODS: Pelves of 6 fresh frozen male cadavers were sectioned as 4 mm. tissue blocks in planes sagittal and perpendicular to the axis of the prostatomembranous urethra from the bladder neck to the bulb of the corpus spongiosum. Sections were photographed and prepared in situ for histological staining (hematoxylin and eosin, Masson's trichrome and phosphotungstic acid hematoxylin). RESULTS: The structure of the male urethral sphincteric complex was demonstrated to include the cylindrical rhabdosphincter surrounding the prostatomembranous urethra and a fascial framework, principally consisting of the ventral subpubic fascia and medial fascia of the levator ani musculature. The histological appearance of the rhabdosphincter at its dorsal aspect suggested a suburethral musculofascial plate. Rhabdosphincteric muscle fibers were oriented in vertical and ventrolateral directions with attachments to the subpubic fascia and the medial fascia of the levator ani. CONCLUSIONS: The structural components and their relationships suggest mechanisms whereby the complex is suspended and stabilized within the deep pelvis, and achieves urethral closure. Our study furthers an understanding of the anatomical basis for male urinary continence and micturition, and is expected to have primary importance in the effort to preserve urinary function following major pelvic surgery.

Adult↗