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Isolation of mycoplasma membranes by digitonin.

The cell membrane of Mycoplasma hominis was isolated by lysing the cells with digitonin. Electron microscopy and chemical, density gradient, and electrophoretic analyses of the membrane proteins showed the membranes so obtained, like those isolated by osmotic lysis, to be relatively free of cytoplasmic contaminants. Sensitivity to digitonin lysis depended on temperature but was not affected by Mg(2+) ions and was only slightly affected by the age of the culture. Accordingly, it seems that digitonin may be used for the isolation of cell membranes from sterol-requiring mycoplasmas that tend to be fairly resistant to osmotic lysis.

Bacterial Proteins↗

Comparison of conventional and immunofluorescent techniques for the detection of Entamoeba histolytica in rectal biopsies.

Two comparative studies of six staining techniques in detection of Entamoeba histolytica in rectal biopsies were performed. The techniques evaluated were direct and indirect fluorescent antibody technique, and four conventional stains: PAS, H & E, Trichrome, and PTAH). On ethanol-fixed tissue, the direct fluorescent antibody technique was the most sensitive, detecting amoebas in 84% of rectal biopsies from 19 aspirate-positive dysentery patients. PAS staining was specific, almost as sensitive, and amoebas could be rapidly detected. The other three conventional methods, however, detected amoebas in only 53--58% of the aspirate-positive patients and required considerable efforts to find the amoebas. The indirect fluorescent antibody technique performed on ethanol-fixed tissues was comparable to the direct technique. The same technique used on formalin-fixed tissues had high background staining, but amoebas could still be detected more efficiently than with conventional staining. Following 20 hr or more treatment with metronidazole, no patient had amoebas detectable in their rectal biopsies by any technique used. Patients with amoebic dysentery and necrotic ulcers on protoscopy had amoebas detected more frequently in rectal biopsy than did patients with mild disease as determined by proctoscopy. Entamoeba histolytica in rectal biopsies can be detected most easily in ethanol-fixed tissue stained with either a fluorescent antibody or with PAS. If amoebas are not detected in the rectal biopsy, amoebic colitis cannot be excluded.

Adult↗

[Determination of contents of lipid peroxidation products in lipoproteins by using precipitation systems].

The impacts exerted by three different systems of precipitation of apo-B-containing lipoproteins, i.e. phosphotungstic acid-Mg2+ heparin-Mn2+ and heparin-Ca2+, on the lipid peroxidation (LPO) parameters in the fraction of high-density lipoproteins (HDLP) and in the aggregate fraction of low and very low density lipoproteins (LDLP, VLDLP) were comparatively analyzed. The systems of phosphotungstic acid-Mg2+ and of heparin-Ca2+ were found to have the comparable results related with the content of LDLP and VLDLP. While, the use of the precipitation system based on heparin-Mn2+ entails overstated LPO parameters in both lipoprotein fractions. It was demonstrated on the basis of all studied precipitation systems that the aggregate LDLP and VLDLP fraction had the highest content of isopropanol-soluble LPO products. The highest indices for the LPO heptane-soluble products were registered in the HDLP.

Apolipoproteins B↗

Interference of levodopa and its metabolites with colorimetry of uric acid.

Reportedly, levodopa (L-DOPA) administration produces spuriously high values for plasma uric acid as measured by the commonly used phosphotungstic acid-hydroxylamine colorimetric method. We confirm this interference, not only by L-DOPA but also by three of its major metabolites: dopamine, 3,4-dihydroxyphenylacetic acid, and 3-methoxy-4-hydroxyphenylacetic acid. However, at therapeutic concentrations in plasma (less than 5 mg/L), the maximum spurious uric acid concentration due to L-DOPA is less than 2 mg/L. Also, at reported peak plasma concentrations of L-DOPA plus three of its major metabolites, the maximum spurious uric acid concentration due to all four compounds combined is less than 8.5 mg/L. Therefore, the hyperuricemia observed with this method in some patients who are chronically receiving L-DOPA cannot be attributed only to interference by L-DOPA and its metabolites in the colorimetric determination of uric acid. Evidently L-DOPA may increase laboratory values for plasma uric acid concentrations, both by pharmacological and chemical mechanisms.

3,4-Dihydroxyphenylacetic Acid↗

Staining of muscle tissue for quantitative studies and automated morphometry.

Seventy-six staining tests were carried out of paraffin sections of human and animal muscle to find a suitable staining method for quantitative morphometry of muscle fibers. The results were evaluated under the light microscope, on black and white photomicrographs and on an image analysing computer, the Quantimet 720.A brilliant scarlet-phosphotungstic acid-tartrazine method is described and recommended for automated morphometry after additional testing on 140 sections of developing human muscle using the Quantiment 720.

Animals↗

Effects of anoxia on the isolated perfused rat heart: histological, histochemical and electron-microscopic observations.

A study to evaluate hematoxylin-basic fuchsin-picric acid (HBFP) and some other conventional stains in the demonstration of early anoxic lesions of the perfused rat heart has been undertaken. The extent of the anoxic injury was readily demonstrable electron-microscopically and in semithin sections by light microscopy, and at 1.5--2 hours practically all myocardial cells showed irreversible changes. The HBFP method was unreliable, giving false positive and false negative results both in the control and anoxic specimens. No clear changes were seen in the four histochemical dehydrogenase reactions applied, whereas damaged myocytes lost some of their affinity to eosin. Contraction bands were frequently seen in the anoxic hearts stained by the phosphotungstic acid-hematoxylin method. However, their presence was not in a linear accordance with the extent of the lesion. The perfusion model is not directly comparable to the myocardial infarct caused, for example, by ligating the coronary artery but, however, the present results suggest the difficulty of demonstrating the very early anoxic myocardial lesion by means other than electron microscopy.

Animals↗

Microthrombi formation after severe head trauma.

This study was undertaken to look for trauma-related fibrinous microthrombi in traumatized human brains. Fifty brains from patients with variable time intervals between trauma and death were fixed in 10% formaldehyde. Sections from the contusioned area and from the corresponding area of the contralateral hemisphere were embedded in paraffin and 50 non-traumatized brains were used as controls. After sectioning and embedding, 10 microns sections were stained with haemalum and eosin (HE) and phosphotungstic acid-hematoxylin (PTAH). Stained fibrinous microthrombi were counted in each hemisphere and in control sections. More microthrombi could be found in the contusioned areas of the brain than in the contralateral side or in control sections.

Adolescent↗

Brain tumors of mixed tissue origin: staining procedures to distinguish glial from connective tissue.

Described is a unique mixed intracerebral tumor composed of schwannoma, piloid astrocytoma, and angiomatous malformation. Review of conventional staining methods for distinguishing glial from connective tissue fibrils reveals that Mallory's phosphotungstic acid-hematoxylin (PTAH) method is less specific than is generally recognized. Knowledge of these pitfalls, combined with use of hematoxylin and eosin stains, and reticulum impregnations are currently most useful in making the distinction by light microscopy. Criteria for malignancy of schwannoma and other mesenchymal tumors should be based on number of mitotic figures and invasiveness rather than on pleomorphiam of cells. Prior knowledge of the location of the tumor may lead to diagnosing some schwannomas as other types of tumor.

Adult↗

Role of Na(+)-H+ exchange on reperfusion related myocardial injury and arrhythmias in an open-chest swine model.

The inhibition of Na(+)-H+ exchange (NHE) with amiloride analogues in vitro has been shown to prevent reperfusion arrhythmias and additional cell necrosis. Inhibition of intracellular Ca2+ overload via NHE inhibition has been suggested as a mechanism of these protective effects. The aim of this study was to examine whether treatment with amiloride analogues reduces the incidence of reperfusion arrhythmias and limits infarct size in vivo. Open-chest swine were exposed to a 30-minute left anterior descending artery (LAD) occlusion and 180 minutes of reperfusion during atrial pacing at 150 ppm. Intravenous 5-(N,N-dimethyl)-amiloride (AML, 5 micrograms/kg per min) was administered in the treatment group (n = 7) and intravenous saline in the control group (n = 7), starting 10 minutes before coronary occlusion. The infusion was continued during ischemia and reperfusion. The area at risk was defined by monastral blue dye and infarct size by triphenyltetrazolium chloride staining. Limb leads ECG and monophasic action potentials (MAPs) from the epicardium in the ischemic area were recorded. There was no significant difference in the size of the area at risk and hemodynamic parameters between the groups. However, the infarcted area was 0.4% +/- 1.0% of the area at risk in the treatment group, whereas it was 62% +/- 29% in the control group (P < 0.05). Pathological examination (Hematoxylin-eosin and Mallory's phosphotungstic acid-hematoxylin staining) revealed that all of the infarcted area consisted of contraction band necrosis. MAP duration in both groups was significantly shortened during ischemia. After reperfusion, MAP duration in the treatment group recovered earlier than that of control group. However, there was no significant difference in the incidence of ventricular tachyarrhythmia between the groups. Inhibition of NHE with AML prevented reperfusion related cell necrosis in the in vivo swine model, but did not reduce the incidence of ventricular tachyarrhythmia.

Action Potentials↗