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PubMed1985

The size and curvature of synapses in the cerebellar cortex of the cat.

At the supra Purkinje layer and the subpial level in the molecular layer of lobules V and VI of the cerebellar cortex of the cat, synaptic profiles were measured in ultrathin serial sections treated with either osmium tetroxide (OsO4) or ethanolic phosphotungstic acid (E-PTA). From trace and chord lengths of the intercepts of synaptic profiles the curvature and the mean caliper diameter of the synapse was calculated. In OsO4-material the curvature of synapses yielded an average angle of about 47 degrees and about 36 degrees in E-PTA material. Although these values are contrary to the assumption of a flat disc, which is commonly required in stereological procedures to estimate caliper diameter, the effect of the curvature on the estimation of the mean caliper diameter is limited. This is shown by serial reconstruction analysis of the largest diameter of synapses from maximal arc and chord length measurements at the subpial and supra Purkinje level. The results provide quantitative data concerning synaptic size, curvature and the frequency of intercepts per synapse at the subpial and supra Purkinje level in the cerebellar cortex in OsO4 and E-PTA material. In addition the advantages and disadvantages of E-PTA and osmiumtetroxide staining in quantitative analysis are discussed.

Animals↗
PubMed1982

Type I collagen segment long spacing banding patterns. Evidence that the alpha 2 chain is in the reference or A position.

Densitometric scans of electron micrographs of type I collagen segment long spacing crystallites stained with uranyl acetate or phosphotungstic acid and uranyl acetate have been correlated with computer-synthesized scans derived from the sequence of the alpha 1(I) and alpha 2 chains. Three models that differ in the location of the alpha 2 chain were used in the computer synthesis; Models A, B, and C have the alpha 2 chain in the A, B, and C chain positions, respectively. For all 13 experimental scans, the order of decreasing correlation with the models was found to be A,B,C. The probability of getting the same order of decreasing correlation all 13 times is 6/6(13). It was also determined at the 0.99 confidence level that the mean of the differences in the correlation coefficients among the models is greater than 0, supporting the conclusion that sequence-derived models best fit the experimental data when the alpha 2 chain is in the A position. Our results also agree with recent studies that show that uranyl ions bind to both positively and negatively charged residues on collagen type I.

Animals↗
PubMed1983

Study of negatively stained images of Sendai virus nucleocapsids using minimum-dose system.

Using minimum-dose system and optical diffraction, effects of electron irradiation on negatively stained images of trypsin-straightened nucleocapsids of Sendai virus were semiquantitatively compared for uranyl-acetate (UA) and phosphotungstic acid (PTA). The results confirmed the superiority of UA in display of fine structures and showed that both UA- and PTA-stained images tended to turn from a one-sided to a two-sided image during irradiation, the general contrast of the picture increased in the UA-stained images but not in the PTA-stained ones, and furthermore the electron doses for the richest information were 18 000 to 30 000 e-/nm2 for UA, but 1000 e-/nm2 for PTA, under the condition used. The optical diffraction patterns of the UA-stained nucleocapsids, its analysis by the superposition method, and rotational harmonics of end-on views of nucleocapsids, together indicated that the most probable arrangement of subunits was 13 per turn of a helix with 5-nm periodicity. This helix also had an arrangement of subunits parallel to the axis. The occurrence of 2.5 nm periodicity was probably produced by an arrangement of a UA-penetrable concave substructure of the subunit.

Capsid↗
PubMed1998

Reference standardization and triglyceride interference of a new homogeneous HDL-cholesterol assay compared with a former chemical precipitation assay.

A homogeneous HDL-c assay (HDL-H), which uses polyethylene glycol-modified enzymes and sulfated alpha-cyclodextrin, was assessed for precision, accuracy, and cholesterol and triglyceride interference. In addition, its analytical performance was compared with that of a phosphotungstic acid (PTA)/MgCl2 precipitation method (HDL-P). Within-run CVs were < or = 1.87%; total CVs were < or = 3.08%. Accuracy was evaluated in fresh normotriglyceridemic sera using the Designated Comparison Method (HDL-H = 1.037 Designated Comparison Method + 4 mg/L; n = 63) and in moderately hypertriglyceridemic sera by using the Reference Method (HDL-H = 1.068 Reference Method - 17 mg/L; n = 41). Mean biases were 4.5% and 2.2%, respectively. In hypertriglyceridemic sera (n = 85), HDL-H concentrations were increasingly positively biased with increasing triglyceride concentrations. The method comparison between HDL-H and HDL-P yielded the following equation: HDL-H = 1.037 HDL-P + 15 mg/L; n = 478. We conclude that HDL-H amply meets the 1998 NCEP recommendations for total error; its precision is superior compared with that of HDL-P, and its average bias remains below +/-5% as long as triglyceride concentrations are < or = 10 g/L and in case of moderate hypercholesterolemia.

Chemical Precipitation↗
PubMed1986

A source of error in the chromatographic study of 35S-sulfate labeled mucous glycoproteins secreted by the gill epithelium of Mytilus edulis.

HPLC combined with [35S]-sulfate/[3H]-glucosamine radiolabeling were employed to study the synthesis and secretion of mucous glycoproteins. The secreted radiolabeled glycoproteins were separated from the medium by precipitation with a mixture of trichloroacetic-phosphotungstic acids (TCA/PTA). The redissolved glycoproteins were chromatographed on an anion exchange protein column at varying pH of the mobile phase and fractions were collected for liquid scintillation counting. Varying the pH of the mobile phase from pH 3 to 7 resulted in a decrease of glycoprotein bound [35S] from 69.5 to 0.5% of the total recovered [35S]-sulfate with the remainder recovered as free [35S]-sulfate. The [3H]-labeled glycoprotein recovered under the uV peaks at this pH range was 99.5%. When high performance size exclusion chromatography was performed the change in mobile phase pH did not affect the 100% recovery of either [35S]-or [3H]-labels under the uV peaks. No free [35S]-sulfate was obtained when [35S]-labeled glycoproteins were separated from the medium using dialysis. These data suggest that the standard method of TCA/PTA precipitation of [35S]-labeled glycoproteins may cleave the [35S]-sulfate ester linkages to the oligosaccharide chains. The [35S]-sulfate may then rebind to the macromolecule by a relatively strong noncovalent bond. This may prove critical in anion exchange protein HPLC studies.

Animals↗
PubMed1980

Ultrastructural localization of basic proteins in cytoplasmic granules of rat eosinophils and mast cells.

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) methods were used to localize basic protein at the ultrastructural level in cytoplasmic granules of rat eosinophils and mast cells isolated using a Metrizamide gradient. Intense reaction was seen in the granules of EPTA-treated eosinophils. Following incubation of the cells for 2 hr in EPTA alone, the matrix was stained. After longer incubation (10 hr), however, both the matrix and core were stained. Cytoplasmic granules of the mast cell show a slight or negative reaction with EPTA. With the AS technique, a large number of silver particles were seen in the nucleus of both eosinophils and mast cells. The mast cell cytoplasmic granules showed intense reaction, while those from eosinophils showed no clear reaction. Acetylation of the cells under conditions sufficient to block most free amino groups prio to EPTA or AS treatment greatly reduced (EPTA) or abolished (AS) the reaction. The results indicate 1) that eosinophil granules contain basic proteins both in the matrix and the core, 2) that the mast cell granules contain a basic protein (probably the alpha-chymotrypsin-like enzyme), which reacts strongly with AS, and 3) that the AS and EPTA methods have different specificities.

Animals↗
PubMed1978

A quantitative electron microscopic study of synapse formation in dispersed cell cultures of rat cerebellum stained either by Os-UL or by E-PTA.

Synapse formation was followed in dispersed cell cultures of rat cerebellums stained either by osmium-uranyl-lead (Os-UL) or by ethanolic phosphotungstic acid (E-PTA). The numerical densities of synapses stained by Os-UL were always significantly higher than those stained by E-PTA (from 3 to 35 days in vitro). This difference suggests that some portion of the populations of both immature and mature synapses was not stained by E-PTA. The width of the synaptic cleft (28.4nm) in synapses stained by E-PTA was only 9nm more than that of the cleft in synapses stained by Os-UL (19.4nm), suggesting that some portion of one or both of the synaptic membranes is stained by E-PTA. Analysis of data from 7 other staining procedures utilizing both ethanolic and aqueous solutions demonstrated that the differences in cleft width described above appear to be due to the various affinities of the stains for different portions of synaptic membranes, and do not represent differences due to shrinkage artifact. In examining the parameters of synaptic structure during development of the cultures, a statistically significant increase in both the height and width of the presynaptic dense projections was found. Changes in synaptic morphology during synaptogenesis in this culture system were similar to those reported for the cerebellar cortex in vivo.

Animals↗
PubMed1997

Evaluation of two homogeneous methods for measuring high-density lipoprotein cholesterol.

We evaluated the performance of two homogeneous assays for quantifying HDL cholesterol (HDL-C) and compared them with the phosphotungstic acid (PTA)/ MgCl2 assay. Both homogeneous HDL-C assays were precise, having a within-run CV of < 1.20% and a between-run CV of < 4.07%. The HDL-C values (y) measured by the two homogeneous methods correlated well with those by the PTA/MgCl2 method (x): y = 1.00x + 64.98 mg/L, r = 0.987, Sy/x = 27.99 mg/L (n = 152) for the polyethylene glycol-modified enzymes/alpha-cyclodextrin sulfate (PEGME) assay (Kyowa), and y = 0.84x + 106.51 mg/L, r = 0.984, Sy/x = 26.10 mg/L (n = 152) for the polyanion-polymer/detergent (PPD) assay (Daiichi). The specificity of the PEGME method seemed better than that of the PPD method, as the PPD method was markedly interfered with by supplemental LDL-C. Addition of 20 g/L triglycerides produced a negative error of approximately 18% in both homogeneous assays. Bilirubin and hemoglobin had little influence on the PEGME method; hemoglobin had little effect on the PPD method. Bilirubin, however, markedly decreased the readings by the PPD method. We found the PEGME assay superior to the PPD assay for routine HDL-C testing, because the PPD assay is relatively inaccurate and not specific.

Bilirubin↗
PubMed1983

Ultrastructural localization of phenothiazines and tetracycline: a new histochemical approach.

To provide high resolution information on the subcellular localization of the phenothiazines and tetracycline, we have developed a new histochemical method that circumvents the difficulties inherent in classical electron microscopic tissue preparatory procedures. Specific and reliable localizations of these drugs were accomplished by their rapid precipitation with phosphotungstic acid (PTA) at pH 7. A cell suspension of Ehrlich ascites carcinoma cells was incubated with a given drug (2.5 x 10(-4) M) and then briefly cross-linked with 1% glutaraldehyde at 4 degrees C. After washing, the cells were exposed to 2% PTA (pH 7) to precipitate the drug at its binding sites. Then the samples were rapidly dehydrates in 80% ethylene glycol (4 degrees C) and embedded in the polyester, Vestopal W. This protocol provides a low denaturation, low extraction approach to tissue preparation. Control samples (without drug) demonstrated an amorphous distribution of PTA throughout the cell and no specific dense precipitates. Those cells treated with the phenothiazines (chlorpromazine or fluphenazine) or tetracycline demonstrated very discrete (4-8 nm), electron-dense drug-PTA reaction products associated with different nuclear components as well as several cytoplasmic organelles. These subcellular localizations verify the binding sites reported by the biochemical literature. In addition, several previously unresolvable binding sites are reported. The rationale and limitations of this procedure are presented. This new histochemical methodology may have broad applications in the study of drug distribution, receptors, and drug-induced pathology and toxicity that may provide new information regarding drug action and design.

Animals↗
PubMed1981

Development of the bovine acrosome. An ultrastructural and cytochemical study.

In the present study the development of the bovine acrosome was investigated using conventional electron-microscopical techniques as well as the phosphotungstic-acid (PTA) technique (Rambourg 1967) including enzymatic digestion experiments. As in other species and in accordance with previous light-microscopical studies (Clermont and Leblond 1955) four phases of acrosomal differentiation can be discerned; the Golgi-phase, cap-phase, acrosome-phase, and maturation-phase. In the bull no internal pattern of the acrosomal content can be observed, either with conventional uranyl acetate-lead citrate staining or with the PTA-techniques. Our results support the observation in other species (Fawcett et al. 1971) that no intrinsic polymerization or crystallization process of the acrosomal content is responsible for acrosomal shaping. Some of our results suggest the influence of external forces on acrosomal development in the bull. During the cap-phase and the acrosome-phase accumulation of smooth endoplasmic reticulum and a layer of fine filaments can be observed in the Sertoli-cell cytoplasm, immediately adjacent to the developing acrosome. A temporary influence of these structures on acrosomal development seems possible. The PTA-positive staining of the developing bovine acrosome is probably due to the presence of acrosomal glycoproteins; however, our results do not exclude the possibility that molecules other than glycoproteins contribute to the positive PTA-staining of the developing acrosome.

Acrosome↗
PubMed1993

"GA-banding": a new terminology and a study of the glutaraldehyde-induced band pattern of type I collagen fibrils.

The negative staining D-band patterns of glutaraldehyde-reacted collagen fibrils were compared to those of fresh collagen fibrils. Negative staining was obtained by using 1% phosphotungstic acid (PTA) diluted in phosphate buffer 0.1 M, pH 7.4. The stain was dripped onto grids where native type I collagen fibrils, isolated from bovine dermis, were collected. Ultrastructural pictures were digitized to form microdensitometric traces. The glutaraldehyde-induced patterns showed fifteen light bands (micrographs) or negative peaks (microdensitograms), whose D-locations were constant and characteristic. In order to make this ultrastructural feature a precise reference parameter, these bands were called "GA-bands" and numbered. When comparing this averaged microdensitogram with that of negatively stained fresh fibrils, peak "GA1" and peak "GA7" were observed to correspond to peak "X2" (known as N-terminal telopeptide region) and peak "X3" (known as C-terminal telopeptide region) respectively, while there was no correspondence between the other peaks of the two traces. It means that the regions where preexistent crosslinks exist are unaffected by interaction with glutaraldehyde, while in the other regions, where new glutaraldehyde-crosslinks occur, the band pattern modifies. The unchanged D-location of peaks "GA1" and "GA7" leads to the conclusion that the D-shortening induced by glutaraldehyde is not due to shifting of tropocollagen molecules but to changes in their orientation with respect to fibril long axis or in secondary-tertiary structure of collagen.

Animals↗
PubMed1971

Elaboration of the matrix glycoprotein of enamel by the secretory ameloblasts of the rat incisor as revealed by radioautography after galactose- 3 H injection.

The elaboration of enamel matrix glycoprotein was investigated in secretory ameloblasts of incisor teeth in 30-40-g rats. To this end, the distribution of glycoprotein was examined histochemically by the use of phosphotungstic acid at low pH, while the formation of glycoprotein was traced radioautographically in animals sacrificed 2.5-30 min after galactose-(3)H injection. Histochemically, the presence of glycoprotein is observed in ameloblasts as well as in the enamel matrix; in ameloblasts glycoprotein occurs within the Golgi apparatus in amounts increasing from the outer to the inner face of the stacks of saccules, and is concentrated in condensing vacuoles and secretory granules; in the enamel matrix, glycoprotein is observed within linear subunits. Radioautographs at 2.5 min after injection demonstrate the uptake of galactose-(3)H label by Golgi saccules, indicating that galactose-(3)H is incorporated into glycoprotein within this organelle. After 5-10 min, the label collects in the condensing vacuoles and secretory granules of the Golgi region. By 20-30 min, the label appears in the secretory granules of the apical (Tomes') processes, as well as in the enamel matrix (next to the distal end of the apical processes, and at the tips of matrix prongs). In conclusion, galactose contributes to the formation of glycoprotein within the Golgi apparatus. The innermost saccules then distribute the completed glycoprotein to condensing vacuoles, which later evolve into secretory granules. These granules rapidly migrate to the apical processes, where they discharge their glycoprotein content to the developing enamel.

Ameloblasts↗
PubMed1996

Chromatin defects in normal and malformed human ejaculated and epididymal spermatozoa: a cytochemical ultrastructural study.

Cytochemical defects in chromatin were examined by transmission electron microscopy (TEM) after the staining by alcoholic phosphotungstic acid (PTA) of normal and malformed ejaculated spermatozoa from 35 male partners of infertile couples, and in six sperm samples retrieved from the caput epididymidis of men affected by obstructive azoospermia. PTA staining was also analysed in normal ejaculates of fertile men after incubation of the washed spermatozoa with dithiothreitol (DTT) to reduce disulfides to thiols, or with DTT followed by iodoacetamide, a blocking agent for thiol groups. PTA stained 63 (27-100)% of malformed heads and 25 (10-100)% of normal sperm heads (median (range) n = 35; P = 0.0001, Wilcoxon matched pairs test). The percentage of normal heads stained by PTA was negatively correlated with the percentage of heads of normal form, with condensed chromatin and a normal acrosome (Spearman r = 0.75; P = 0.0001), and positively correlated with the percentage of malformed heads after conventional TEM analysis (Spearman r 0.60; P = 0.0001). Staining with PTA in normal heads was not correlated with the presence of non-condensed chromatin in otherwise normal sperm heads evaluated by conventional TEM analysis. In spermatozoa recovered from the caput epididymidis, 15% of normal heads were stained with PTA, significantly fewer than in ejaculated sperm samples (P = 0.014). The reduction of disulfides to thiols was associated with PTA staining of all normal heads, and this was prevented by incubation with iodoacetamide. We conclude that PTA staining of the nuclei of human ejaculated spermatozoa may indicate a defect of chromatin condensation, owing to an excess of free thiol groups. The lower percentage of normal epididymal sperm heads that stained with PTA in cases of obstructive azoospermia compared with ejaculated sperm may be related to an overoxidation of thils owing to the ageing of spermatozoa.

Chromatin↗
PubMed2001

Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) has a pathogenesis distinct from other forms of human prion disease: disease-related prion protein (PrP(Sc)) is readily detectable in lymphoreticular tissues. Quantitation of risk of secondary transmission, and targeting of risk reduction strategies, is limited by lack of knowledge about relative prion titres in these and other peripheral tissues, the unknown prevalence of preclinical vCJD, and a transmission barrier which limits the sensitivity of bioassay. We aimed to improve immunoblotting methods for high sensitivity detection of PrP(Sc) to investigate the distribution of PrP(Sc) in a range of vCJD tissues. METHODS: We obtained tissues at necropsy from four patients with neuropathologically confirmed vCJD and from individuals without neurological disease. Tissues were analysed by sodium phosphotungstic acid precipitation of PrP(Sc) and western blotting using high sensitivity enhanced chemiluminescence. FINDINGS: We could reliably detect PrP(Sc) in the equivalent of 50 nL 10% vCJD brain homogenate, with a maximum limit of detection equivalent to 5 nl. PrP(Sc) could be detected in tissue homogenates when present at concentrations 10(4)-10(5) fold lower than those reported in brain. Tonsil, spleen, and lymph node were uniformly positive for PrP(Sc) at concentrations in the range of 0.1-15% of those found in brain: the highest concentrations were consistently seen in tonsil. PrP(Sc) was readily detected in the retina and proximal optic nerve of vCJD eye at levels of 2.5 and 25%, respectively of those found in brain. Other peripheral tissues studied were negative for PrP(Sc) with the exception of low concentrations in rectum, adrenal gland, and thymus from a single patient with vCJD. vCJD appendix and blood (Buffy coat fraction) were negative for PrP(Sc) at this level of assay sensitivity. INTERPRETATION: We have developed a highly sensitive immunoblot method for detection of PrP(Sc) in vCJD tissues that can be used to provide an upper limit on PrP(Sc) concentrations in peripheral tissues, including blood, to inform risk assessment models. Rectal and other gastrointestinal tissues should be further investigated to assess risk of iatrogenic transmission via biopsy instruments. Ophthalmic surgical instruments used in procedures involving optic nerve and the posterior segment of the eye, in particular the retina, might represent a potential risk for iatrogenic transmission of vCJD. Tonsil is the tissue of choice for diagnostic biopsy and for population screening of surgical tissues to assess prevalence of preclinical vCJD infection within the UK and other populations.

Animals↗
PubMed1975

Ultrastructural and biochemical observations on interphase nuclei isolated from chicken erythrocytes.

Adult hen erythrocyte nuclei are isolated from cells or haemolysed in situ by acting on the plasma membrane with rotating knives or with non-ionic detergents. When the isolation medium contains magnesium ions (1 mM), sucrose (0-4 M) and Tris buffer (0.01 M, pH 7-5) called SMTOG (see text), the ultrastructure in thin sections through the condensed chromatin bodies, after staining with either uranyl-lead or phosphotungstic acid (PTA), is similar to that found in the intact cell. Hence it can be concluded that the 2 phases which comprise chromatin, the o- and e-phase, survive nuclear isolation. These are so called because the structural units in chromatin are arranged at the surface of the nucleus into one or more layers and give rise to oddly (o) and evenly (e) numbered bands. The 0-phase is also largely retained after extensive washing in 0-07 M NaC1 as shown by electron microscopy and biochemical measurements; only 6% of the total nuclear protein is removed, a value small compared with the fractional amount of the chromatin protein calculated to lie in the o-phase, about 70%. After extensive washing in saline-EDTA there are structural changes in chromatin, but biochemical data show that the molecules in the o-phase are also largely retained; loss of protein amounts to between 5 and 11%. These data suggest that the o-phase is a structural component of the chromatin bodies. They support the hypothesis that condensed chromatin is formed by folding superunit threads. These units consist of a central thread-like element about 17 nm diameter which stains preferentially with uranyl-lead and forms the e-phase, with an outer cylindrical shell forming the o-phase of total diameter about 28nm. The 5-10% proteins removed by salt washes are located exclusively in a particulate component, quite likely the chromatin. They have been examined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. There are about 10 or more protein species, ranging in molecular weight from 21000 upwards. The groups of large granules previously found in the nuclear sap of intact erythrocytes are shown to be associated with an amorphous or finely fibrillar body.

Animals↗
PubMed1976

A fine structural and E-PTA study of photoreceptor synaptogenesis in the chick retina.

Photoreceptor synaptogenesis in the embryonic and hatchling chick retina was studied with conventional EM techniques and ethanolic phosphotungstic acid (E-PTA). The photoreceptors line up between 11 and 13 embryonic days with their undifferentiated synaptic bases facing the outer plexiform layer (OPL). E-PTA staining at 11 embryonic days does not reveal any para-membranous specializations of the receptors but numerous stained punctae adhaerentes are observed in the OPL. At 13 embryonic days neurites of presumed bipolar and horizontal neurons are aligned parallel to the bases of the receptors and cytoplasmic protrusions of the receptors project between some of these neurites to form dyad appositions. An osmiophilic undercoating, which is not E-PTA positive at this time, is present on the cytoplasmic face of the receptor membrane in these apposition regions. Between 13 and 15 embryonic days the filopodial protrusions of the receptors continue to elongate further and become aligned with neurites in dyad and triad appositions. The osmiophilic undercoating now extends along the entire inner surface of the receptor pedicle protrusions and becomes E-PTA positive. Between 15 and 17 embryonic days focal aggregations of osmiophilic and E-PTA stained material appear along the membranes of the protrusions and there is some E-PTA staining of the postsynaptic densities and intervening cleft material. Between 17 and 21 embryonic days mature ribbon synapses are observed on the surfaces of the conical-shaped, receptor pedicles where the ribbons and their synaptic vesicles are associated with the dense aggregations (arciform densities), seen earlier as isolated focal aggregations, and the receptor undercoating is restricted to non-synaptic regions. E-PTA staining shows that ribbons are positively stained around their borders only and that they are contiguous with the intensely stained arciform densities. The cleft material and postsynaptic densities of some synapses first stain as V-shaped junctions and later as Y-shaped junctions. These observations suggest that ribbon synaptic junction formation begins with an alignment of pre- and postsynaptic membranes and the presence of the receptor presynaptic membrane undercoating, followed by the appearance of the presynaptic arciform densities and some staining of the cleft material and postsynaptic densities. These events are followed by the appearance of synaptic ribbons which are associated with the presynaptic arciform densities and by a further differentiation of the cleft material and postsynaptic densities.

Animals↗
PubMed1987

On the heterogeneous glycosylation of the membranes of the trans Golgi network in rabbit luteal cells.

In rabbit luteal cells the transmost element (G2) of the Golgi apparatus bears cytochemical resemblances to the limiting membrane of lysosomes and it was suggested that lysosomal membranes may originate from the above element. But in the normal Golgi apparatus it cannot be made out whether the considered molecules are indeed membrane bound. Perfusing the rabbit ovary with buffer containing monensin or ammonium chloride allowed to vesiculate the trans Golgi network (G2-G1) selectively. Controls showed a well-preserved ultrastructure. Parts of the limiting membrane of the vacuoles derived from the transmost reticulum (G2) were spiny coated and carried an osmiophilic inner layer. They also showed a heavy precipitate for acid phosphatase (AcPase) and were strongly stained with phosphotungstic acid (PTA) at low pH. By neutralizing the acidic groups, involved in the PTA-staining, it was possible to show that the same membranes were more heavily glycosylated. The MvB's and the limiting membrane of lysosomes showed the same staining characteristics. The other membrane domains revealed a gradient in PTA staining and in AcPase activity. It is concluded that the trans Golgi network (G2-G1) is an acidic compartment. The presence of differentially glycosylated membranes reveals a sorting mechanism for membranous components. The highly glycosylated membrane stretches seem to be involved in endocytosis and in the formation of lysosomal membranes.

Acid Phosphatase↗
PubMedDate not supplied

Suicidal poisoning by endothall.

A fatal case of suicidal ingestion of the herbicide endothall (7-oxabicyclo [2,2,1]heptane-2,3-dicarboxylic acid) is described. Endothall was determined after initial phosphotungstic acid deproteination followed by extraction using 1,2-dichloroethane:tert-butanol (2:1) and subsequent injection into the gas chromatograph equipped with a flame ionization detector. Identity was confirmed by thin-layer chromatography, infrared spectrophotometry, and gas chromatography/mass spectrometry.

Adult↗