Microincineration and elemental X-ray microanalysis of single Bacillus cereus T spores

Single whole spores of bacillus cereus T were analyzed by scanning electron microscopy and electron microprobe X-ray microanalysis before and after high-temperature (600 degrees C) ashing in air. High-temperature ashing consisted of a centripetal oxidation of the spore surface combined with pyrolysis of the spore's interior. Ashing of single spores produced a compact central ash particle, mimicking the much larger unashed spore body in outline but containing craterlike microregions, and a peripheral thin ash film. Ashing mostly eliminated the spore's organic matrix; however, ash residues still gave residual carbon-characteristic X-ray counts. Ashing of single spores produced a two-, five-, and six-fold increase of potassium, magnesium, and calcium X-ray intensities, respectively. Iron, although low in actual counts, became detectable after ashing. Phosphorus characteristic X-rays were decreased by 41% after ashing, while volatilization lowered sodium and manganese X-ray intensities by over 80%. High-temperature ashing enhanced element-characteristic X-ray intensities of the non-volatilizable mineral(ized) elements of spores by compacting them into ash residues, more so than by simply abolishing their organic matrix. Microincineration appears a generally useful preconcentration technique for elemental detection and localization in X-ray microanalysis.     

Post-mortem elemental redistribution in rat studied by X-ray microanalysis and electron microscopy

Summary Post-mortem elemental redistribution in various tissues from rat was studied by means of electron probe X-ray microanalysis, and correlated with morphological changes in these tissues. Pancreas, liver and cardiac muscle were removed from the animal either immediately, or after some hours after death. Elemental distribution at the cellular level was studied by X-ray microanalysis of thick cryosections. Calcium redistribution at the subcellular level was studied using tissue fixed with glutaraldehyde/oxalate. In all tissues, post-mortem redistribution of electrolytes had taken place within 2 h. The cellular concentrations of Na, Cl and Ca increased markedly, those of Mg and K decreased; no significant changes were found in the concentrations of P and S. The number of oxalate precipitates (indicating the presence of calcium) increased both in the mitochondria and in the cytoplasm and endoplasmic reticulum, reaching a maximum at 2 h. Morphological changes included mitochondrial swelling and vesiculation of the endoplasmic reticulum. Since the post-mortem ion shifts are similar to those encountered in some diseases and types of cell injury, great care has to be taken in the interpretation of X-ray microanalytical results from autopsy material.     

Abnormal calcium distribution in human parathyroid adenomas as possible cause of primary hyperparathyroidism

Current knowledge suggests that normal parathyroid glands and parathyroid adenomas have different sensitivities to environmental calcium. In search for morphological equivalents, 5 normal human and 10 porcine parathyroid glands, as well as 10 human parathyroid adenomas were investigated with regard to intracellular and extracellular calcium distribution. The glands were incubated for 2, 4, 6 and 20 h in tissue cultures using HAM's F10 medium with various calcium concentrations. For visualization of the calcium distribution in the tissue the method of pyroantimonate precipitation was applied. Specificity of the reaction was controlled by X-ray microanalysis. Shifts of the calcium pyroantimonate precipitates were quantitated by morphometry using an area-counting system. The results demonstrate that in normal parathyroid glands calcium precipitates are distributed randomly. Incubation of normal glands in medium with low calcium concentration (0.6 mM) provoked reduced amounts of intracellular and extracellular calcium complexes. When the incubations were performed in medium with high calcium content (2.6 mM), calcium accumulated inside parathyroid and stroma cells. In contrast to normal parathyroid glands, parathyroid adenomas fixed immediately after surgery showed an atypical calcium distribution with low amounts of intracellular and high amounts of extracellular calcium grains. The data suggest that in normal parathyroid glands the intracellular calcium concentration follows the extracellular environmental calcium concentration. Thus, calcium modulates parathyroid hormone (PTH) secretion via intracellular regulatory mechanisms. In parathyroid adenomas the calcium transport via the tumor cell membrane appears to be disturbed resulting in lowered intracellular calcium levels. This is remarkable since the environmental calcium concentration is elevated due to the hypercalcemia of primary hyperparathyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis

Recent data suggest that changes in ionic content, primarily potassium, play a pivotal role in the progression of apoptosis. However, the changes in total element content, i.e., sodium (Na), magnesium (Mg), phosphorous (P), chlorine (Cl), potassium (K), and calcium (Ca), during apoptosis have not been evaluated. Electron probe X-ray microanalysis (EPXMA) was used to measure total element content in U937 cells before and after the induction of apoptosis. As an experimental model we used U937 cells irradiated with ultraviolet (UV) light. Apoptosis was evaluated with phase-contrast microscopy, with scanning and transmission electron microscopy, and with the fluorescent dye bisbenzimide (Hoechst 33342). Plasma membrane permeability as a measure of cell death was determined by trypan blue dye exclusion. To investigate element content with EPXMA, cells were cryoprepared, i.e., cryofixed and freeze-dried, and analyzed as whole cells using a scanning electron microscope. We found that the UV irradiation induced rapid (within 2 h) morphological changes associated with apoptosis, such as plasma membrane blebbing, condensation of the chromatin, and the formation of membrane-bound apoptotic bodies. At this time, 95% of the apoptotic cells excluded trypan blue dye. EPXMA results demonstrated that UV light-irradiated apoptotic cells (cells with membrane-bound apoptotic bodies) had a lower Cl content (P < 0.001) and K content (P < 0.001) and a higher Na content (P < 0.001) in comparison with nonirradiated control cells. Also, P and Ca content was higher in apoptotic cells than in control cells, but this difference did not reach statistical significance. No differences were found in Mg. These data indicated that morphological changes characteristic of apoptotic cell death are related with significant changes in sodium, chlorine, and potassium content. In addition, we demonstrated that these changes in elemental composition were not associated with loss of cell membrane integrity.     

Electron probe X-ray microanalysis of human muscle biopsies

Summary The elemental composition of human muscle fibres have been determined by electron probe microanalysis. In order to distinguish between different types of fibres, two approaches were used. In one approach individual fibres were isolated, portions of them used for a typing by histochemical methods and the main part used for X-ray microanalysis. In the other approach the muscle biopsy was serial-sectioned, some sections used for a histochemical typing and the others (16 m thick cryosections) used for X-ray microanalysis in the electron microscope.The comparison of the ratios between P, S and K in Study No. 1 and 2 indicates different concentrations of sulphur in the subsarcolemmal zone and in the interior of the fibre. Both routes give information on all elements (except the ten lightest ones) contained in the fibres or in sections of them, provided the concentration is high enough. In order to obtain quantitative data, expressed as mmol/kg_dw, the spectra of the specimens were compared to those of standards of known composition and the data subjected to a so called ZAF-correction (corrections for the atomic number effect, absorption of X-rays in the specimen and secondary fluorescence). Quantitative data concerning phosphorus, sulphur, chlorine and potassium were obtained in Study No. 2. A significantly higher sulphur concentration was found in type IIA muscle fibres as compared to those of type I.     

Morphology of healthy human parathyroid glands in cytologic smears

OBJECTIVE: To compare the morphology of different parenchymal cell types in healthy human parathyroid glands (HPGs) in cytologic smears with their structure on histologic sections and to establish criteria for their recognition in smears. STUDY DESIGN: The HPGs of 47 subjects (27 females and 20 males) were incidentally removed during surgery on the thyroid gland. The tissue of glands with a normal macroscopic and microscopic appearance was analyzed in cytologic smears and on histologic sections. RESULTS: In cytologic smears (as well on histologic sections), dark and light chief cells predominated. Dark chief cells, which were more numerous than light ones, had a smaller nucleus without a visible nucleolus. The nucleus of light chief cells was larger with 1 or 2 nucleoli visible. The cytoplasm of both types of chief cells was poorly defined, grey-blue and often vacuolated. In the smears, the cytoplasm of oxyphilic cells was dense, gray-rose and well defined. In dark oxyphilic cells, the nuclei were smaller and without a visible nucleolus. Light oxyphilic cells had a larger nucleus and visible nucleolus. CONCLUSION: The identification and distribution of 4 parenchymal cell subtypes in the smears of pathologically altered HPGs may yield insights into the possible role of these cells in a specific disease.     

Normal responsiveness of serum parathyroid hormone to beta-adrenergic blockade in patients with secondary hyperparathyroidism

Infusion of calcium gluconate (15 mg Ca++/kg body weight in 4 h) to 6 patients with secondary hyperparathyroidism (due to mild renal insufficiency) decreased serum parathyroid hormone (PTH) levels to the same degree (on a percent basis) as in normal subjects. Serum PTH values at 4 h were 60 +/- 4.5 (SEM)% of baseline in the patients and 59 +/- 2.9% of baseline in the normal subjects. Infusion of propranolol (1 mg i.v. bolus followed by an infusion of 60 micrograms/min for 2 h) to 7 additional patients with secondary hyperparathyroidism also decreased serum PTH to the same degree as in normal subjects. Serum PTH values at 2 h were 68 +/- 10.4% of baseline in the patients and 68 +/- 3.3% of baseline in the normal subjects. The studies indicate normal responsiveness of serum PTH to calcium or beta-adrenergic blockade in secondary hyperparathyroidism due to mild renal insufficiency.     

DNA ploidy pattern of parathyroid parenchymal cells in renal secondary hyperparathyroidism with relapse.

The nuclei of parathyroid parenchymal cells, analyzed using image cytometry (ICM), in relapsing and non-relapsing secondary hyperparathyroidism due to uremia, showed a DNA-distribution pattern of diploid type. Nevertheless, some differences were observed within the groups, as regards the concept of 'scattered cells' in ICM DNA histograms. The relative incidence of 'scattered cells' was particularly high in the histograms from parathyroid glands with nodular hyperplasia and in those from parathyroid parenchyma grafted into the skeletal musculature. In these two kinds of parathyroid specimens, the 'scattered cells' were both of chief-cell and oxyphil-cell types. In contrast, 'scattered cells' were not so conspicuous when parenchymal cells of glands with diffuse hyperplasia were analyzed. As there is some clinical and histopathological evidence that the cells in both nodular-hyperplastic and autografted parathyroid parenchyma have increased growth potential, it is hypothesized that the relative incidence of the 'scattered cells' in the ICM DNA histograms indicates an increased proliferative activity.     

Cohort study on effects of parathyroid surgery on multiple outcomes in primary hyperparathyroidism

Objectives To assess the effects of surgery compared with conservative treatment (no surgery) for primary hyperparathyroidism.Design Cohort study.Setting Nationwide Danish cohort.Participants 3213 patients, mean age 61 (SD 16) years, with a diagnosis of primary hyperparathyroidism between 1980 and 1999. 1934 (60%) underwent surgery and 1279 (40%) were treated conservatively.Main outcome measures Occurrence of fractures, osteoporosis, kidney or urinary tract stones, acute myocardial infarction, angina pectoris, cardiac arrhythmias, arterial hypertension, heart failure, stroke, acute pancreatitis, stomach or duodenal ulcers, muscle pain, malignant diseases, psychiatric disorders, and mortality.Results At diagnosis of primary hyperparathyroidism, patients who subsequently underwent surgery had a lower prevalence of previous fracture (odds ratio 0.64, 95% confidence interval 0.51 to 0.80), acute myocardial infarction (0.59, 0.42 to 0.83), stroke (0.57, 0.37 to 0.88), psychiatric disorders (0.54, 0.31 to 0.94), and painful muscle disorders (0.44, 0.26 to 0.76), whereas kidney stones (2.49, 1.93 to 3.23) and acute pancreatitis (2.77, 1.33 to 5.76) were more prevalent. After diagnosis, the risks of fractures (hazards ratio 0.69, 0.56 to 0.84) and gastric ulcers (0.59, 0.41 to 0.84) were lower in patients treated surgically than those treated conservatively. Events involving kidney or urinary tact stones were more prevalent in patients treated surgically than patients treated conservatively (1.87, 1.30 to 2.68). Mortality was lower in patients treated surgically (0.65, 0.57 to 0.73).Conclusions Patients treated surgically for primary hyperparathyroidism have a lower prevalence of fractures and gastric ulcers than patients treated conservatively. The type of treatment had no effect on the occurrence of cardiovascular events.     

Ultrasonic examination of the parathyroid glands, bone x-ray and calcium deposits of the eye in diagnosis of secondary hyperparathyroidism in light of 12 observed cases]

Twelve patients with chronic renal failure treated with repeated dialysis underwent subtotal parathyroidectomy. Histologic examination revealed hyperplasia parathyroidal adenoma in all patients. Diagnostic correlation between bone X-ray, ultrasound of parathyroid glands, calcium deposits in cornea and conjunctiva, and histological findings has been analysed. Radiological abnormalities have been noted in 8 patients (66%), parathyroid glands hyperplasia in 9 (75%), and corneal and conjunctival calcium deposits in 10 (83%) patients. These data confirm the value of techniques under study for the diagnosis of the secondary hyperparathyroidism in patients with chronic renal failure.     

Parvalbumin is expressed in normal and pathological human parathyroid glands.

The parathyroid glands are of major importance in calcium homeostasis. Small changes in the plasma calcium (Ca2+) concentration induce rapid changes in parathyroid hormone (PTH) secretion to maintain the extracellular Ca2+ levels within the physiological range. Extracellular Ca2+ concentration is continuously measured by a G-protein-coupled Ca2+-sensing receptor, which influences the expression and secretion of PTH. The mechanism of signal transduction from receptor sensing to PTH secretion is not well understood, but changes in PTH secretion are tightly linked to changes in the cytosolic Ca2+ concentration. Using immunohistochemistry and Western blot analysis, we detected the EF Ca2+ binding protein parvalbumin (PV) in normal and in hyperplastic and adenomatous human parathyroid glands. The strongest PV signal was present in chief cells and water clear cells, whereas in oxyphilic cells only a weak signal was observed. Immunohistochemistry and in situ hybridization of the PTH indicated a co-localization of PV and PTH in the same cell types. Because changes in the cytosolic Ca2+ concentration are believed to influence the process of PTH secretion, a possible role of PV as a modulator of this Ca2+ signaling is envisaged.