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PubMed1994

Correlations between amino acid hydrophobicity scales and stain exclusion capacity of type 1 collagen fibrils.

The relationship between the negative staining band pattern of type 1 native collagen fibrils and the amino acid distribution along the fibril axis was studied by comparing averaged microdensitograms with theoretical traces calculated on the basis of different amino acid parameters. As well as the spatial parameter "bulkiness" (volume/length, ratio), various literature-reported scales of "hydrophobicity" were tested. Two "hydrophobicity" sets allowed a better fit with the actual patterns than "bulkiness" values. However, a general improvement in simulations was achieved by associating most "hydrophobicity" sets with the "bulkiness" set. These results suggest that amino acid "hydrophobicity" plays a key role in the appearance of negative staining patterns but a composite mechanism would seem to occur: the accessibility of available intermolecular interstices may be conditioned by molecular hindrance, corresponding to amino acid "bulkiness" as well as by water-repulsion effect, which correlates with amino acid "hydrophobicity." Moreover, a detailed comparison of actual and simulated patterns suggests that a modulation exists in the effectiveness of these two factors along each D-period according to the different molecular packing and concentration of hydrophobic amino acid clusters within overlap regions and gap regions, respectively.

Amino Acids↗
PubMed1989

[Correlation of vessel invasion and bone metastasis in prostatic adenocarcinoma. A clinico-pathological and immunohistochemical study of 33 cases of total prostatectomy].

Thirty three cases of prostatic adenocarcinoma treated by total perineal prostatectomy were studied clinicopathologically and immunohistochemically. There were 17 patients with clinical stage B, 12 with stage C and 8 with stage D. Interrelationship of tumor grade, surgical local tumor extent, vessel invasion, perineural invasion and bone metastasis was examined. For the identification of the vessel invasion, Ulex europaeus agglutinin 1 was adopted immunohistochemically. Tumor grade and local tumor extent were respectively correlated with bone metastasis. Vessel invasion was correlated with local tumor extent. Eight of 18 cases (44%) with vessel invasion and none of 15 cases without vessel invasion had bone metastasis. Although correlated with grade, perineural invasion had no significant effect on bone metastasis. The investigation of tumor staining used by monoclonal antibody for prostatic acid phosphatase (PSAP) disclosed that negative stained cases were associated with lower grade tumors and that one of 14 positive stained cases (7%) and 7 of 19 negative stained cases (36%) had bone metastasis. We concluded that vessel invasion may be a new prognostic pathological parameter and that monoclonal PSAP staining is also a useful method to predict malignant potential of prostatic cancer.

Acid Phosphatase↗
PubMed1983

Immunofluorescence studies on epidermal keratinization in some skin disorders with keratin antisera.

The immunofluorescence technique using antisera against human plantar stratum corneum fibrous protein (total keratin) and 64K M.W. keratin subunit isolated from total keratin by SDS polyacrylamide gel electrophoresis (PAGE) was used to examine epidermal keratinization in some skin disorders. In normal skin, total keratin was distributed throughout the epidermis, while 64K keratin was localized at the suprabasal layers. In the case of squamous cell carcinoma (SCC), the tumor cells were uniformly stained positive with total keratin antiserum, whereas diminished or negatively stained cells were observed with 64K keratin antiserum (64K antiserum), suggesting that the tumor included cells in various stages of differentiation. In basal cell epithelioma (BCE), most of the tumor cells were negatively stained with 64K antiserum being consistent with the histologic observation that BCE is originated from the basal cells. However, some of the tumor cells were stained positive with 64K antiserum, indicating that individual cell keratinization might occur in BCE. In lichen planus, an inflammatory disease demonstrating so-called lichnoid tissue reaction, positively stained colloid bodies in the upper dermis were observed either with total keratin antiserum or with 64K antiserum. It was suggested that colloid bodies resulted from individual keratinization of damaged keratinocytes during inflammation.

Basal Cell Carcinoma↗
PubMed1993

Immunohistochemical staining for thyroglobulin in poorly differentiated carcinoma of the thyroid.

Paraffin sections of 14 specimens of poorly differentiated tumours of the thyroid were stained for thyroglobulin using polyclonal antibodies and a streptavidin-biotin peroxidase staining system. The better differentiated areas showed positive staining, while the poorly differentiated areas gave weakly positive or negative staining. Thyroglobulin staining is useful in attributing a thyroid epithelial cell origin to a tumour, but negative staining does not exclude this.

Carcinoma↗
PubMed1967

Electron microscopy of a strain of Bordetella bronchiseptica.

A strain of Bordetella bronchiseptica that had been isolated from a rat hepatoma cell culture was investigated by means of electron microscopy. Bacteria were examined after (i) negative staining with phosphotungstate or uranyl acetate, (ii) metal shadowing with platinum-palladium, and (iii) fixation with glutaraldehyde followed by embedding, sectioning, and staining. The multilayered bacterial cell walls appeared lobulated in negatively stained and in metal-shadowed specimens; the lobules were demarcated by grooves, 100 to 200 A in width, but without interruption of continuity in any layer of the cell wall. Cross sections of fixed material revealed wrinkled cell walls in many-but not all-preparations. Bacterial cell membranes and cytoplasm were similar to those of other gram-negative bacilli (e.g., Escherichia coli). Bacteria fixed in 1.5% glutaraldehyde contained intertwined or whorled fibrils, down to about 20 A in thickness. The flagella were peritrichous, measured about 200 A in width, and were composed of braided strands, about 20 A in width.

Animals↗
PubMed1995

Hepatitis C virus within a malignant lymphoma lesion in the course of type II mixed cryoglobulinemia.

Hepatitis C virus (HCV) has been implicated as the major etiologic factor sustaining B-cell clonal expansion in type II mixed cryoglobulinemia (MC). A putative pathogenetic role of HCV in the development of MC-associated B-cell malignancies has also been speculated. We report for the first time the localization of HCV within a parotid non-Hodgkin's lymphoma (NHL) lesion in the course of HCV-related type II essential MC, an important step to implicate any infectious agent in the lymphomagenesis. Plus and minus strand HCV RNA was first demonstrated by polymerase chain reaction on the whole RNA from the lesion. Further immunohistochemical studies localized HCV c22 proteins in the residual ductal or acinar parotid structures, which also abnormally expressed HLA-DR antigens. Weak c22 signals were inconstantly detected in cells strictly confined around the residual epithelium, while all the remaining infiltrating cells in the parotid lesion stained c-22-negative. Staining for c33 and c100 HCV antigens was negative. In situ hybridization (ISH) studies again identified the residual parotid epithelial cells as the site of HCV infection and replication in the NHL lesion. Sialotropic viruses previously involved in lymphoproliferation, ie, Epstein-Barr virus and human herpesvirus-6, were absent in the same tissue lesion. According to the current models of B-cell lymphomagenesis, a role of HCV as an exogenous antigenic stimulus should be considered for NHL development in the present case, whereas malignant B cells do not appear permissive of active HCV replication. Further efforts would be worthwhile to clarify a role of HCV infection in the development of some B-cell malignancies.

Aged↗
PubMed2000

Prognostic significance of cyclin D1 protein levels in early-stage larynx cancer treated with primary radiation.

Recent laboratory experiments have demonstrated that cyclin D1 levels (cycD1) can influence radiosensitivity. The purpose of the current study is to evaluate the prognostic significance of cycD1 for local recurrence in early-stage larynx cancer treated with primary radiation therapy. The study was conducted using a matched case-control design in 60 early-stage (T1-T2/N0) larynx cancer patients. All patients had squamous cell carcinoma of the larynx and were treated with primary radiation to a total median dose of 66 Gy in daily fractions of 2 Gy, without surgery or chemotherapy. Thirty patients who suffered a local relapse in the larynx after treatment served as the index case population. These 30 cases were matched by age, sex, site (glottic vs. supraglottic), radiation therapy technique/dose, and follow-up, to 30 control patients who did not experience a local relapse. Immunohistochemical staining from cycD1 was performed on the paraffin-embedded specimens. The pathologist, blinded to the clinical information, scored each of the specimens on a four-point intensity scale (0 = no stain, 1 = faint, 2 = moderate, 3 = strong) and percent distribution. Patients were considered to be positive for cyclin D1 if the staining was 2+ or greater with a percent distribution of at least 5%. By design of the study, the two groups were evenly balanced with respect to age, sex, stage, radiation dose, and follow-up. CycD1 levels correlated with proliferating cell nuclear antigen levels. Low levels of cycD1 significantly correlated with local relapse; 19/30 (63%) of the index cases stained negative, while only 10/30 (33%) of the control cases stained negative (P = 0.03). These data suggest that low levels of cycD1 correlate with relatively radioresistant early-stage larynx carcinoma. With larger more confirmatory clinical and laboratory data, this data may have significant clinical implications. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 22-28 (2000).

Carcinoma, Squamous Cell↗
PubMed1985

The effect of beta-adrenoceptor blocking agents on evolving myocardial necrosis in coronary ligated rats with and without reperfusion.

The left coronary artery of rats was ligated either permanently, or for a period of 40 or 60 min, with subsequent reperfusion. In experiments with permanent occlusion, the hearts were removed and investigated 5 h after the coronary ligation, or immediately after death in animals which died earlier. The hearts from the reperfusion experiments were investigated 60 min after reopening the occluded artery. The extent of the ischaemic and necrotic areas of the hearts was determined. A quantitative photometric method was developed for this purpose, using "negative staining" with Evans blue for the ischaemic area, and "negative staining" with triphenyltetrazolium chloride for the necrotic area. In experiments in which ligation was permanent, the percentage of the ischaemic area which underwent necrosis increased with the time after coronary occlusion. In reperfusion experiments, myocardial necrosis was detected earlier than in experiments with permanent coronary ligation. The beta-adrenoceptor blocking agents pindolol, propranolol, and metoprolol significantly decreased the percentage of necrosis in experiments with permanent ligation of the coronary artery. The most selective of the beta-adrenoceptor blockers, i.e. metoprolol was tested in the reperfusion experiments. In these experiments, the amount of necrosis was also significantly decreased.

Adrenergic beta-Antagonists↗
PubMed1988

Three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus. A flagellin lacking the outer domain and its amino acid sequence lacking an internal segment.

We obtained a three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus CB15 from electron micrographs of negatively stained preparations. The C. crescentus filament appears, both in negative stain and in the frozen-hydrated state, significantly smoother and narrower than other filaments. Its helical symmetry, and unit cell size, however, are similar to that of other filaments. Although the molecular weight of the C. crescentus flagellin is about half that of other plain flagellins, there is only one monomer per unit cell as indicated by diffraction studies and by linear mass density measurements with the scanning transmission electron microscope. Alignment of the primary amino acid sequences of Salmonella typhimurium (serotype i) and C. crescentus (29,000 Mr) flagellins shows that whereas there is homology at the amino and carboxyterminal ends of the two sequences, the central segment of the S. typhimurium sequence has no homology to that of C. crescentus. A correlated comparison between the three-dimensional reconstructions of the two filaments and primary amino acid sequences of the two flagellins suggests that: (1) the C. crescentus subunit is missing the outer molecular domain but is, otherwise, similar to that of S. typhimurium; (2) the outer molecular domain in S. typhimurium corresponds, therefore, to a central stretch of the primary amino acid sequence; and (3) the outer molecular domain, missing in C. crescentus, is not obligatory for flagellar motility.

Amino Acid Sequence↗
PubMed1978

Comparison of rhapidosomes and asbestos microfibrils.

Rhapidosomers (cylindrical nucleoprotein rods of bacterial origin) show great structural similarity to the microfibrils of chrysotile asbestos when negatively stained and observed with the electron microscope. If the negative stain is omitted, the asbestos retains its structural detail whereas the rhapidosomes appear to be unstructured bodies. When the microscope is adjusted into a selected area diffraction mode, the asbestos shows characteristic electron diffraction patterns whereas the rhapidosomes appear to be amorphous to electron diffraction.

Asbestos↗
PubMed1989

Basal-body-associated disks are additional structural elements of the flagellar apparatus isolated from Wolinella succinogenes.

The intact flagella of Wolinella succinogenes, a gram-negative, anaerobic bacterium with a single polar flagellum, were obtained by an improved procedure, introduced recently by Aizawa et al. (S.-J. Aizawa, G. E. Dean, C. J. Jones, R. M. Macnab, and S. Yamaguchi, J. Bacteriol. 161:836-849, 1985) for the flagellum of Salmonella typhimurium. Disks with a diameter of 130 +/- 30 nm, which were attached to the basal body of the isolated intact flagella, could be identified by electron microscopy as additional structural elements of the bacterial flagellar apparatus. In freeze-dried and metal-shadowed samples, two rings of the basal body were detected on one side and a terminal knob was located on the other side of the disks. Suspension of the flagellar apparatus in acidic solution dissociated the flagellar filaments, yielding hook-basal body complexes with and without the associated disks. If whole cells were subjected to low pH, double disks of the same diameter and with a central hole of about 13 nm could be isolated. Similar parallel disks could be seen also in negatively stained whole cells. When uranyl acetate was used for negative staining of the intact flagella, concentric rings were detected on the disks, similar to the concentric membrane rings found by Coulton and Murray (J. W. Coulton and R. G. E. Murray, J. Bacteriol. 136:1037-1049, 1978) on platelike arrays of proteins in outer membrane preparations of Aquaspirillum serpens. Because the disks of W. succinogenes can be isolated together with the flagellar hook-basal body complex, they appear to be basal-body-rather than secondary membrane-associated structures. It is possible that these disks are the bearing or stator of this rotary device.

Cell Fractionation↗
PubMed1985

Inhibiting effect of dexamethasone on evolving myocardial necrosis in coronary-ligated rats, with and without reperfusion.

Previous findings suggested that glucocorticosteroids may be able to preserve ischemic myocardial tissue from cellular necrosis. In the present investigation, the left coronary artery in rats was ligated either permanently, or reperfusion was allowed after an occlusion time of 40 min. After 5 h of permanent coronary ligation, or after 60 min of reperfusion, respectively, the hearts were investigated as to the extent of their ischemic and necrotic areas. The ischemic areas were determined by 'negative staining' with Evans blue, and the necrotic areas by 'negative staining' with triphenyltetrazolium chloride. Dexamethasone orthophosphate in both kinds of experiments significantly decreased the percentage of the ischemic area which had undergone necrosis.

Animals↗
PubMed1980

Molecular sieve in renal glomerular and tubular basement membranes as revealed by electron microscopy.

Bovine glomerular basement membrane (GBM) was isolated and purified according to a modification of Spiro's method. Rat and bovine tubular basement membranes (TBM) were isolated and purified by sonic disruption or by the method of Carlson et al. Electron microscopic studies on the ultrastructure of GBM and TBM were performed after negative staining with 1% phosphotungstic acid solution, pH 7.3. When negatively stained, GBM and TBM were seen as fragments varying in size. The surface of the membranes showed a characteristic felt-like or spongy appearance. At higher magnification, GBM and TBM showed a fine meshwork composed of strands and pores which three-dimensionally resembled a crystal lattice. Pores were fairly uniform in size and shape. They were round, oval or polygonal in shape. Some of the pores were elongated to form short straight or bent channels. Strands were also uniform in diameter and surrounded a pore or channel. For an average of 50 pores, the long dimension was 3.1 +/- 0.6 nm and the short dimension 2.5 +/- 0.3 nm in bovine GBM, 3.8 +/- 1.2 and 2.5 +/- 0.7 nm in bovine TBM, and 4.9 +/- 1.5 and 2.8 +/- 0.6 nm in rat TBM, respectively. The strand was 1.8 +/- 0.3 nm in diameter in bovine GBM, 2.5 +/- 0.6 nm in bovine TBM and 3.7 +/- 0.7 nm in rat TBM for an average of 50 strands. The diameters of the pores were less than or close to the short axis of an albumin molecule. It was concluded that renal GBM and TBM were molecular sieves composed of pores and strands.

Animals↗
PubMed1994

A new case of malignant mesothelioma of the tunica vaginalis testis. Immunohistochemistry in comparison with an adenomatoid tumor of the testis.

Malignant mesothelioma of the tunica vaginalis testis is an extremely rare tumor with 41 previously reported cases. The histological and immunohistological features of a new case in an 80-year-old patient are described and compared with an adenomatoid tumor of the tunica vaginalis testis, which is considered to be the benign variant of malignant mesothelioma. Both tumors revealed strong cytoplasmic staining for a panepithelial antibody (Lu-5) and membranous staining for BMA-120 (a mesothelial/endothelial cell marker) but yielded negative staining results with the endothelial cell markers QBend-10 (CD 34), Factor VIII-related antigen (vWF) and UEA-1. There was also negative staining for CEA, Ber-EP4, HEA-125 and Blood group related antigens A, B, H. An identical staining pattern was evident in normal and reactive mesothelial cells. Our data support a mesothelial rather than an endothelial derivation of the adenomatoid tumor studied.

Aged↗
PubMedDate not supplied

Apoptosis, cell proliferation and expression of oncogenes in gastric carcinomas induced by preoperative administration of 5-fluorouracil.

The purpose of this study was to examine the correlations among enhancement of apoptosis, cell proliferation and expression of oncogenes in gastric carcinomas induced by preoperative oral administration of 5-fluorouracil (5-FU). The occurrence of spontaneous apoptotic cell death in 42 patients with gastric carcinoma was analyzed in the biopsy specimens preoperatively. p53 status was examined by polymerase chain reaction-single strand confirmation polymorphism and sequencing. Fourteen patients received oral administration of 5-FU at 300 mg/body/day for 7 days preoperatively. For detection of apoptotic cells, apoptotic incidences (AIs) were examined by the terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate biotin nick end labeling method, on gastric carcinoma lesions based on the endoscopic findings before administration in the biopsy and resected tissues. Expressions of p53, Bcl-2, Bax gene and proliferating cell nuclear antigen (PCNA) were also examined by immunohistochemical staining. On preoperative biopsy, p53 point mutation was observed in 14 of the 42 tumors. The immunohistochemical staining status and point mutation of p53 gene (positive or negative) were identical in 32 of the 42 tumors (76.2%). The average AIs of the biopsy specimens were 1.58+/-1.26% on p53-negative staining (n=19) and 1.14+/-1.02% on p53-positive staining (n=23), a significant association was not recognized between p53 expression and AI. In the preoperative administration group, the PCNA labeling index was significantly higher in the biopsy specimens than in the resected tissues (43. 6+/-12.8% vs. 35.3+/-8.8%, p<0.01). In addition, postoperatively, the rate of AI was significantly more accelerated in p53-negative staining (n=6) than in p53-positive staining (n=8) (0.89+/-0. 65%right curved arrow 4.18+/-3.26%, p<0.05 vs. 1.20+/-0.60%right curved arrow 2.60+/-2.60%, NS). There was no significant correlation between AI and Bcl-2 or Bax staining. Immunohistochemical analysis of p53 and PCNA stainings in biopsy specimens appears to be a well-characterized indicator of sensitivity of chemotherapy in gastric carcinomas.

Apoptosis↗
PubMed1990

Features of the aperiodic microfibrils associated with mouse dental basement membrane demonstrated by ultrastructural histochemistry.

Histochemical features of aperiodic microfibrils (AMF) in mouse tooth germs were examined at the electron microscopic level. Intact and EDTA-isolated materials obtained from one day old first molars were used for ruthenium red (RR) staining, ferritin permeability, periodic acid-silver methenamine (PAM) impregnation, fibronectin localisation, negative staining on cryo-sections and tannic acid fixation. Electron microscopy and negative staining demonstrated that AMF traverse the basal lamina and penetrate below the inner enamel epithelium. In addition to RR staining, PAM impregnation and tannic acid fixation showed deposition on the AMF which was associated with basal laminae. RR staining and tannic acid fixation also indicated the presence of glycoprotein-rich materials in the lamina lucida. The AMF were derived from the lamina lucida which was closely associated with tannic acid-positive granular materials. The precipitation of silver particles by PAM impregnation was seen on banded collagen fibrils, basal lamina and AMF, but the staining features of AMF differed distinctly from those of collagen fibrils. The distribution of ferritin particles revealed that the basal lamina covering EDTA-isolated papilla tissue is a continuous structure. Immuno-reactions for fibronectin were detected on the basal lamina and AMF. Our results suggest that AMF are derived from the glycoprotein-rich lamina lucida and that their histochemical characteristics resemble those of basal lamina.

Animals↗
PubMed1994

Immunolocalization of tropomodulin, tropomyosin and actin in spread human erythrocyte skeletons.

The human erythrocyte membrane skeleton consists of a network of short actin filaments cross-linked into a hexagonal network by long, flexible spectrin molecules. The lengths of the short actin filaments (33 +/- 5 nm) at the central junctions are proposed to be stabilized and limited by association with tropomyosin and the tropomyosin-binding protein, tropomodulin. Here, we use immunogold labelling followed by negative staining to specifically localize tropomodulin, tropomyosin and actin to the sites of the central junctions in spread membrane skeletons. In addition to negative staining, immunogold labelling for tropomodulin at the sites of the central junctions was also visualized by a quick-freeze, deep-etch, rotary-replication technique. These experiments confirm previous indirect evidence that the short filaments at the central junctions are indeed actin filaments and provide the first direct evidence that tropomodulin and tropomyosin are associated with the erythrocyte actin filaments in situ.

Actins↗
PubMed1976

Herpes encephalitis. Rapid diagnosis and treatment with antiviral drugs.

The use of specific IgM antibodies and direct electron-microscopic examination of brain biopsies or vesicle fluid was tested as means of raped diagnosis in 6 cases of herpes simplex encephalitis seen consecutively in Montreal. In 2 of 3 biopsies herpes viruses were seen by negative staining of a cell extract within 1 hr. In the negative case, the biopsy was done almost 1 month after onset. In 2 additional cases herpes virus particles were found directly in the fluid of isolated vesicles. In the last 2 cases, who survived, the diagnosis of herpes encephalitis rested upon the demonstration of a greater than 4-fold rise in complement fixing herpes simplex virus antibodies in convalescent sera and upon the appearance late in the course of the encephalitis of specific antibodies in the cerebrospinal fluid. The early appearance of specific IgM antibodies contributed to the diagnosis in 4 of the 6 cases. Antiviral therapy was attempted in alternate cases (3 cases) but was not successful. Brain biopsy is rarely performed for diagnostic purposes but when prompt antiviral therapy is contemplated, the examination of the biopsy material for herpes virus particles by electron microscopy in negative staining and thin sections can rapidly and reliably confirm the diagnosis.

Aged↗