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PubMed1983

Cytophotometrical histochemical method of simultaneous measurement of two ingredients (DNA, proteins, enzymes) in the same cell.

There are 2 alternative procedures for cytophotometrical determination of 2 histochemical ingredients (DNA, proteins, and enzymes) in the same cell. The 1st possibility consists of the sequential measurement of 2 ingredients, when the 2nd chromophore is revealed after the determination and extraction of the 1st chromophore. Another one is the simultaneous measurement, when the 2 histochemical reactions (at 1st with the 1st chromophore and then with the other one) are performed on the fixed sections. So each of the sections includes 2 chromophores simultaneously. Next after photometry in 2 wavelengths, the quantity of each chromophore is calculated. This method gives a characteristic of mutual distribution of 2 ingredients in one and the same cell by coefficient of correlation. This article accentuates the causes of the limitations, and the estimation of mistakes. In the appendix there are described methodics and programmes of calculations.

Acetylcholinesterase↗
PubMed1985

A new system to consider varying section thickness in histophotometry.

The reference material, an exposed and developed x-ray film, was cutted together with the tissue specimen. By scanning microscope photometry, the mean optical density of the silver layer was measured. By this way, variations in section thickness are to be considered for histophotometrical investigations. A good proportionality between the optical density of the silver grain layer and the relative thickness of the clear film carrier, estimated by microinterferometry, was proved.

Histological Techniques↗
PubMed1996

Complications during transurethral vaporization of the prostate.

OBJECTIVES: To review adverse events during transurethral vaporization of the prostate (TVP) and to compare them with those occurring during transurethral resection of the prostate (TURP). METHODS: One experienced surgeon performed 26 consecutive TVPs and 100 TURPs. We monitored blood loss by photometry, fluid absorption by the ethanol method, and hemodynamic stability with a blood pressure manometer. RESULTS: Total blood loss and the rate of blood loss were significantly lower during TVP. Fluid absorption occurred during most TVPs, but it was mostly small volumes. No sudden drop in arterial pressure was recorded during TVP, but this did occur in 11% of the TURPs. Complications that would normally require medical attention (blood loss greater than 800 mL, fluid absorption greater than 500 mL, or hemodynamic instability) were more infrequent during TVP than during TURP (P = 0.012). CONCLUSIONS: Transurethral vaporization of the prostate was associated with fewer intraoperative complications than was TURP.

Aged↗
PubMed2000

Clinical outcome 1 year after transurethral vaporization and resection of the prostate.

OBJECTIVES: To evaluate the 1-year safety and effectiveness of transurethral vaporization of the prostate (TUVP) compared with transurethral resection of the prostate (TURP) in alleviating outflow obstruction. METHODS: Two experienced surgeons performed 26 TUVPs and 28 TURPs. The intraoperative blood loss was measured by photometry and fluid absorption by the ethanol method. The treatments were evaluated by means of the International Prostate Symptom Score (IPSS), quality-of-life score, transrectal ultrasound, prostate-specific antigen level, urinary flow rate, and postvoid residual urine volume. RESULTS: After TUVP, the median IPSS decreased from 22 to 4.5 and the quality-of-life score from 4.5 to 1.5. The corresponding data for TURP were from 25 to 5.5 and from 4.0 to 1.0. The median urinary flow rate increased from 4 to 10 mL/s after TUVP and from 2 to 11 mL/s after TURP. The postvoid residual urine volume decreased to 35% (TUVP) and 15% (TURP) of the preoperative volume. The blood loss was larger during TURP (P <0.04), but complications during follow-up were more frequent after TUVP (P <0.02). Patients with fluid absorption during surgery had a lower quality-of-life score at follow-up (P <0.02) and tended to have a smaller reduction in prostate size (to 79%) than those without absorption (to 67% of baseline). CONCLUSIONS: TUVP and TURP were both effective in alleviating outflow obstruction, but the outcome appeared to be slightly better for TURP.

Aged↗
PubMed1996

Involvement of mitochondria in intracellular calcium sequestration by rat gonadotropes.

Intracellular Ca2+ ([Ca2+]i) dynamics were studied in identified rat gonadotropes using the whole-cell patch-clamp technique in conjunction with Indo-1 photometry. The kinetics of depolarization-induced [Ca2+]i transients vary with Ca2+ load. In addition to a rapid initial decay, large (> 500 nM) [Ca2+]i transients have a slow plateau phase. Application of the mitochondrial inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly slows the decay of [Ca2+]i transients, consistent with stopping uptake of Ca2+ by mitochondria. CCCP causes a small increase of [Ca2+]i at rest. After a large Ca2+ entry the amount is much larger, consistent with release from a mitochondrial Ca2+ pool that fills during cytoplasmic Ca2+ loading. The rate of Ca2+ uptake by mitochondria is dependent upon [Ca2+]i. Consistent with previous studies, gonadotropin releasing hormone (GnRH) induces [Ca2+]i oscillations. The mitochondrial inhibitors CCCP and cyanide (CN-) terminate these oscillations. The mitochondrial ATP-synthase inhibitor oligomycin reduces the frequency and increases the amplitude of the oscillations. In the presence of ruthenium red (a non-specific blocker of the mitochondrial Ca(2+)-uniporter) in the pipette, GnRH does not induce rhythmic [Ca2+]i oscillations. We suggest that mitochondria play a significant role in the rapid clearance of cytosolic Ca2+ loads in gonadotropes and participate in GnRH-induced periodic [Ca2+]i oscillations.

Animals↗
PubMed2000

Inflammation after sclerocorneal versus clear corneal tunnel phacoemulsification.

OBJECTIVE: To compare the postoperative inflammation after phacoemulsification followed by intraocular lens (IOL) implantation by means of sclerocorneal versus clear corneal tunnel incision. DESIGN: Randomized controlled clinical trial. PARTICIPANTS: One hundred eyes of 100 patients were examined at a German University eye hospital. INTERVENTION: One hundred eyes with cataract necessitating phacoemulsification with posterior chamber IOL implantation were randomly assigned to receive a temporal sclerocorneal or clear corneal tunnel incision by a single surgeon. MAIN OUTCOME MEASURES: Preoperative and postoperative inflammation was evaluated by measurement of flare using laser flare photometry. Statistical inference was mainly based on nonparametric group comparisons by use of two sample Wilcoxon tests. RESULTS: Mean anterior chamber flare in the group with sclerocorneal tunnel increased from 7.5 photon counts/ms preoperatively to 19.6 at 6 hours postoperatively and decreased to 11.1 (day 1), 11.7 (day 2), 11.6 (day 3), and 9.2 (5 months) during the postoperative course. The mean flare in the clear corneal tunnel incision group increased from 7.7 preoperatively to 12.9 at 6 hours postoperatively and then decreased to 9.2 (day 1), 9.8 (day 2), 9.1 (day 3), and 9.2 (5 months). Individual postoperative flare changes were significantly lower in the clear corneal tunnel group at the day of surgery (P<0.0001), as well as at day 1 (P = 0.0011), day 2 (P = 0.0079), and day 3 (P = 0.0020). After 5 months, no statistically significant difference was found. CONCLUSIONS: After phacoemulsification and foldable IOL implantation, postoperative alteration in the blood-aqueous barrier was statistically significantly lower with the clear corneal tunnel incision group compared with the sclerocorneal incision group, in the first 3 days postoperatively.

Adult↗
PubMed2000

Adenosine triphosphate mobilizes cytosolic calcium and modulates ionic currents in mouse taste receptor cells.

By using photometry and the patch clamp technique, we identified P(2Y)-like receptors in mouse taste receptor cells (TRCs) and found them to be coupled to Ca(2+) mobilization and ionic current modulation. Particularly, adenosine triphosphate (ATP) and the P(2Y) agonist 2-methylthio-ATP increased intracellular Ca(2+) by stimulating the phosphoinositide pathway, whereas beta, gamma-methylene-D-ATP, a P(2X) agonist, was ineffective. In a distinctive TRC subpopulation, ATP closed Ca(2+) channels. This regulation may underlie the negative feedback tuning neurotransmitter release. By mobilizing intracellular Ca(2+), ATP activated Ca(2+)-dependent Cl(-) channels, the intracellular event that may universally occur upon taste stimulation triggering IP(3) formation and Ca(2+) release in the TRC cytoplasm.

Adenosine Triphosphate↗
PubMed2000

Lidocaine inhibits potassium efflux and hemolysis in erythrocytes during oxidative stress in vitro.

Lidocaine is a widely used local anesthetic agent. The aim of this work was to study the action of lidocaine on human red blood cells exposed to an oxidative stress in vitro. Blood was obtained from healthy volunteers. After separation from plasma, the erythrocytes were suspended in phosphate buffer. Oxidative stress was induced by incubation with a free radical generator, the 2,2' azobis (2-amidinopropane) hydrochloride (AAPH). Erythrocytes were incubated with or without lidocaine at two concentrations (36.93 and 73.85 microM) and with or without AAPH (20 mM). Electron paramagnetic resonance (EPR) spectroscopy was performed to identify the free radical species generated by AAPH using the spin trap 5-5'-dimethyl-L-pyroline-N-oxide (DMPO). Different sets of experiments were run. Potassium efflux was measured by flame photometry in each group at time 0 min and every 30 min of the experiment for 2 h. Hemolysis was studied by the Drabkin method at increasing concentrations of AAPH (20, 50, and 100 mM) and with or without lidocaine (36.93 microM). The oxygen radical absorbance capacity (ORAC) was measured by using allophycocyanin (APC) as a fluorescent indicator protein, and the antioxidant capacity of lidocaine (36.93 microM) was studied by the analysis of fluorescence of the APC. AAPH was shown to produce alkoxyl free radicals. Oxidative stress induced a marked increase in the potassium efflux and the hemolysis that was AAPH dose-dependent. Lidocaine inhibited the potassium efflux and delayed the occurrence of hemolysis. Lidocaine did not show any antioxidant properties for the free radical species generated by AAPH. In this model, lidocaine protects erythrocytes against oxidative stress. This effect is not explained by a free radical scavenging property. The results may be of great interest in clinical practice such as intravenous regional anesthesia or the prevention of ischemia-reperfusion injury.

Amidines↗
PubMed1999

Nitric oxide synthase neurons in different areas of normal aged and Alzheimer's brains.

This study investigated the distribution of nitric oxide synthase-containing neurons in the cerebral cortex of individuals with Alzheimer's disease, and compared them with age-matched controls. Paraffin-embedded sections of the frontal (area 10), occipital (area 17) and entorhinal cortices (area 28), and hippocampal formation obtained from 13 autopsy cases were used in the study. Neurons expressing nitric oxide synthase messenger RNA and protein were identified, respectively, by in situ hybridization and immunohistochemistry. Optical densities of nitric oxide synthase-positive neurons were assessed in 50 randomly selected fields of each of the above regions of the cortices, in each case by microscopic photometry. In the frontal cortex of the Alzheimer group, while a decrease in the number of nitric oxide synthase-positive neurons was evident, the nitric oxide synthase neurons, on the other hand, showed an increased optical density in layers II-IV when compared with those of normal ageing. In the occipital cortices, no significant differences in optical density were recorded between the normal ageing and Alzheimer specimens. In the entorhinal cortex, the optical densities of nitric oxide synthase neurons were again similar between the Alzheimer and age-matched control groups. In the hippocampar formation itself, there was an increase of nitric oxide synthase staining in the Alzheimer patients. These results show that (i) nitric oxide synthase neurons are abundant in the human cortex, (ii) the distribution of nitric oxide synthase neurons differs between different cortical regions, and (iii) there are differences between normal ageing and Alzheimer patients in the frontal cortex and the hippocampus.

Aged↗
PubMed1988

Flow cytometric analysis of stemline heterogeneity.

The stemline heterogeneity of malignant tumors is closely connected with tumor aneuploidy. Both features can be characterized by either chromosome analysis or DNA cytophotometry. DNA analysis may be performed either by single cell photometry or by flow cytometry, of which the respective advantages and drawbacks are presented. Preliminary results of flow cytometric DNA analysis in malignant neoplasms are discussed; the possibilities of multiparametric measurement for quantitative analysis of various biochemical and antigenic properties in DNA stemlines are considered.

Aneuploidy↗
PubMed2000

Analysis of amines in plant materials.

Biogenic amines are conveniently divided into aliphatic monoamines, aliphatic di- and polyamines and aromatic amines. These compounds are shown to fulfill an array of roles in cellular metabolism. Thus, amines are needed for growth and development and their metabolism appears to be coordinated with the cell cycle. Di- and polyamines, among which are putrescine, spermidine and spermine, are ubiquitous polycationic molecules that occur in all living cells. However, plants accumulate a number of specific related compounds under free or conjugated forms. In plant tissues, the molecular diversity combined with the fact that amine contents are highly responsive to developmental and environmental signals encouraged analysts to develop specific procedures for their isolation and characterization. The main goals were to develop high performance routine procedures in terms of selectivity, repeatability and detectability with minimum running costs. Domains of application concern not only fundamental aspects of amine biochemistry and physiology in plants but also increasing needs in the control of food and beverage quality from plant origin. The present review reports the most recent advances in extraction, identification and quantitation of amines in plant tissues with special interest in the analysis of original and uncommon metabolites. Emphasis is directed towards chromatographic and electrophoretic separation methodologies and new detection technologies of both derivatized and underivatized compounds including photometry, fluorometry, amperometry and mass spectrometry.

Amines↗
PubMed2001

On-line deproteinization by adsorption of proteins on a polyethylene powder pre-column for the determination of Na, K, Mg and Ca in human serum by high-performance liquid chromatography.

Deproteinization of human serum was carried out on-line using a polyethylene powder cartridge as pre-column. The serum sample, after dilution, was injected into the chromatograph, and when passing through the cartridge the proteins were adsorbed by the polyethylene. A protein-free eluate was carried to the analytical column while the pre-column was washed with methanol and water to elute the adsorbed proteins by changing the pump channels. After washing, the pre-column was conditioned with the eluent to receive the next sample. Deproteinization was evaluated off- and on-line using pooled serum, and testing the protein residue after passing through a polyethylene cartridge with the Comassie Brilliant Blue reaction. Parameters such as serum volume, eluent and washing solution were investigated. Sodium, potassium, magnesium and calcium determinations were performed by high-performance liquid chromatography with conductimetric detection. The proposed method is suitable for the determination of these cations in serum samples without further treatments. The total analysis time was about 20 min and a linear range from 0.3 to 1.2 mg/l for sodium, potassium, magnesium and calcium was observed. The method was characterized by a precision of about 95% and recoveries from spiked samples were between 96 and 102%. The results for serum samples analysed by the proposed method were compared with photometry and atomic absorption spectrometry.

Adsorption↗
PubMed1989

High-performance liquid chromatographic determination of some polar phospholipids in serum.

The experimental conditions have been optimized for high-performance liquid chromatographic determination of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM) in serum. The phospholipids are separated on a silica gel column, using a mobile phase of acetonitrile-methanol-water (100:10:18, v/v), with ultraviolet photometric detection at 200 nm. The limit of detection was 0.2 micrograms (in 20 microliters) for natural phospholipids and 2.5 micrograms for synthetic phospholipids; the relative standard deviation was ca. 5%. An alternative detection is tensammetry at a mercury electrode, at a potential of -1.8 V, with an a.c. current frequency of 60 Hz and an amplitude of 20 mV. The tensammetric detection has an advantage in its independence of the structure of the phospholipids. In measurements without a column (flow-injection analysis), the tensammetric detection also yields a somewhat lower limit of detection than photometry (0.15 micrograms per 20 microliters), but this value increases more than ten times in chromatographic detection. The precision is poorer and is more susceptible to interferences. The method was applied to the determination of the above substances in the blood of obese children, as a function of physical stress and spa treatment. It was shown that physical exercise causes a decrease in the contents of PE and PC in the patients. On the other hand, the spa treatment has no pronounced effect on the phospholipid content in the blood.

Adolescent↗
PubMed1999

Capillary zone electrophoresis of potassium in human vitreous humour: validation of a new method.

The analysis of potassium in the vitreous humour has long been regarded as an important tool in medicolegal and forensic toxicological investigation, particularly for the determination of the post-mortem interval. The present work was aimed at the optimisation and validation of a reliable, simple and fast capillary electrophoresis method for potassium analysis in the human vitreous humour with indirect UV detection at a wavelength of 214 nm. Electrophoretic separations were carried out in a running buffer comprising 5 mM imidazole, 5 mM 18-crown-6 ether and 6 mM D,L alpha-hydroxybutyric acid (HIBA), adjusted to pH 4.5. Constant voltage runs were carried out by applying a voltage of 500 V/cm at 25 degrees C. The samples were injected in the hydrodynamic mode at the anodic end of the capillary (0.5 p.s.i. for 10 s; 1 p.s.i. = 6894.76 Pa). The method showed good linearity in the concentration range from 6.5 mM to 16.25 microM, with an r2 value of 0.9994. The limit of detection, based on a signal-to-noise ratio of three, was 9.0 microM. Absolute intra-day RSDs of migration times were <0.40%, while the day-to-day values were < or =1.72%. Absolute peak area reproducibility was always better than 2.50%. A comparison of capillary electrophoresis with flame photometry on twelve real autopsy samples showed an excellent correlation with an r2 value of 0.9333. A preliminary application to real cases (20 subjects) was carried out plotting vitreous humour potassium vs. post-mortem interval with a resulting r2 of 0.904 and a Y-intercept of 4.75 mM, in agreement with the existing literature.

Aqueous Humor↗
PubMed2000

Toxicological investigations of the effects carboxymethylcellulose on ciliary beat frequency of human nasal epithelial cells in primary suspension culture and in vivo on rabbit nasal mucosa.

The objective of this study was to investigate the safety of a mucoadhesive carboxymethylcellulose (CMC) formulation for intranasal administration of apomorphine. The effect of different concentrations of CMC on ciliary beat frequency (CBF) was studied using a human nasal epithelial suspension cell culture system. The CBF was determined by computerized microscope photometry. The in vivo rabbit nasal mucosal tolerance of the mucoadhesive polymer was investigated using light microscopy. Twice daily, six rabbits received CMC powder in one nostril and CMC/apomorphine powder in the alternate nostril for 4 weeks. Two control rabbits received air puffs in one nostril and nothing in the alternate nostril. The rabbits were subsequently sacrificed and the stained nasal sections examined microscopically. CMC showed both concentration- and time-dependent inhibitory effects on the CBF. Only mild-to-moderate cilio-inhibition was recorded with the different concentrations of the polymer. CMC (both with and without apomorphine) caused mild-to-moderate inflammation after 4 weeks. Necrosis, squamous metaplasia or ciliary degeneration was not observed. Based on: (1) the mild-to-moderate cilio-inhibition induced by different concentrations of CMC; and (2) the mild-to-moderate nasal mucosal inflammation caused by CMC with and without apomorphine, we conclude that this polymer can be considered as a safe carrier for short-term intranasal administration. However, further investigations are required for its use in the treatment of chronic diseases such as with apomorphine in Parkinson's disease.

Animals↗
PubMed2003

Safety-assessment of 3-methoxyquercetin as an antirhinoviral compound for nasal application: effect on ciliary beat frequency.

It has been shown that 5,7,3',4'-tetrahydroxy-3-O-methylflavone (3-MQ) exhibits antipicornaviral activity. In order to explore the potential of 3-MQ as an antirhinoviral compound for nasal application, the effect of 3-MQ on the ciliary beat frequency (CBF) of human nasal epithelial cells was studied in vitro in the absence or presence of solubility/absorption enhancers (hydroxypropyl-beta-cyclodextrin (HP-beta-CD) or polysorbate 80). Nasal epithelial cells were obtained by protease digestion of surgical specimens of human nasal polyps, and used at confluency. The effect of 3-MQ (2, 10, and 20 microg/ml), HP-beta-CD (1, 3, and 10% (w/v)), polysorbate 80 (0.1, 0.3, and 1% (w/v)), and of the combination of 3-MQ with 3% HP-beta-CD or 1% polysorbate 80, on the CBF was determined by computerized microscope photometry 15 min after incubation with the test compounds; recovery was determined 35 min after rinsing. HP-beta-CD at 1 and 3% did not affect CBF; a reversible decrease (by 37%) was observed at 10%. Polysorbate 80 caused a reversible cilio-inhibitory effect of 40, 53, and 49% at 0.1, 0.3, and 1%, respectively. At 2 and 10 microg/ml, 3-MQ showed a reversible cilio-stimulatory effect of 18 and 14%, respectively. Combined with 3% HP-beta-CD, the reversible cilio-stimulatory effect of 2 microg/ml 3-MQ was preserved, while 10 and 20 microg/ml 3-MQ did not affect the CBF. The combination of polysorbate 80 (1%) and 3-MQ decreased the CBF, which could be attributed to the presence of polysorbate 80. In conclusion, no ciliotoxic effect could be observed for 3-MQ (up to 20 microg/ml) in the absence or presence of HP-beta-CD (3%). The potential of this combination as an antirhinoviral formulation for nasal application will be further explored.

Administration, Intranasal↗
PubMed2000

Safety assessment of selected cyclodextrins - effect on ciliary activity using a human cell suspension culture model exhibiting in vitro ciliogenesis.

The objective of this study was to assess the cilio-inhibitory effect of a series of cyclodextrins using a human cell suspension culture system exhibiting in vitro ciliogenesis. Enzymatically released human nasal epithelial cells were cultured as sequential monolayer-suspension culture showing in vitro ciliogenesis. Ciliary beat frequency (CBF) was determined by computerized microscope photometry. Among the cyclodextrins investigated (gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin, anionic-beta-cyclodextrin polymer, dimethyl-beta-cyclodextrin and alpha-cyclodextrin), it was shown that after 30 min of exposure, gamma-cyclodextrin (10% w/v), hydroxypropyl-beta-cyclodextrin (10.0% w/v) and anionic-beta-CD polymer (8.0% w/v) were not significantly cilio-inhibitory (P0.05). Similarly, CBF remained stable upon cell exposure to alpha-cyclodextrin (2.0% w/v) and dimethyl-beta-cyclodextrin (1.0% w/v). However, higher concentrations of alpha-cyclodextrin and dimethyl-beta-cyclodextrin resulted in mild to severe cilio-inhibition after 45 min of exposure. The effect of alpha-cyclodextrin (5.0% w/v; 54+/-4% cilio-inhibition) was partially reversible while dimethyl-beta-cyclodextrin (10% w/v; 36+/-4% cilio-inhibition) was irreversible. The cilio-inhibition observed in this model was lower than reported for chicken trachea model. Given the fact that (1) irreversible cilio-inhibition observed in this study occurred only at concentrations exceeding those used in pharmaceutical formulations and/or at an unusual exposure time (45 min) and that (2) in an in vivo situation, dilution and mucociliary clearance contribute to further decrease in local concentrations of the applied compound, the results of this study confirm the safety of the cyclodextrins investigated as nasal absorption enhancers.

Cells, Cultured↗
PubMed1984

Effects of glycerol on the inner ear fluid electrolytes of guinea pigs--oral and intravenous administration.

Under sodium pentobarbital anesthesia (25-35 mg/kg), serum, CSF and inner ear fluids from scala tympani perilymph, scala vestibuli perilymph, and scala media endolymph were collected from normal guinea pigs after the oral administration of glycerol (50%, 12 ml/kg) or the intravenous injection of glycerol (1.0 ml/kg). The sodium and potassium concentrations were assessed by microflame photometry. The electrolyte dynamics of the inner ear fluids were compared after these two routes of administration. Serum: There was no significant change in Na or K levels after either route of administration. CSF: The Na concentration increased rapidly after both intravenous and oral administrations of glycerol and remained high until the end of the experiment. The K concentration did not change significantly after intravenous injection. Scala tympani perilymph: The Na concentration increased transiently, after intravenous glycerol, while after oral administration it increased slowly and steadily. The K concentration increased only after oral administration. Scala vestibuli perilymph: The Na level increased slowly and steadily only after oral administration of glycerol, and the K level increased slightly after intravenous injection. Scala media endolymph: No changes in the K level occurred after either route of administration. After oral administration the Na level increased. These experiments show that dehydration persists longer after oral than after intravenous administration of glycerol. In the diagnosis of Meniere's disease, oral administration of glycerol is more effective than intravenous injection.

Administration, Oral↗