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PubMedDate not supplied

[Species specific motility patterns of hyperactivated mammalian spermatozoa and quantitative analysis of the hyperactivation of bull spermatozoa].

Movement tracks of spermatozoa of human, lion, tiger, cow, pig and sheep are recorded by dark field photography (fluid layer thickness 16.7 microns, exposure time 1 s). Comparison before and after capacitation by an incubation of 2 h in modified tissue culture medium TCM 199 with 10% fetal calf serum resulted in two quite different patterns of hyperactivated spermatozoa: 1) Tracks are broadened due to enlarged lateral head displacement or radius of rotating head movements respectively (tiger, lion) and show beside that a markedly increase in erratic motility (human). 2) In the studied species of artiodactyla, cow, pig and sheep, a qualitative new, panicle-like pattern arised as a result of superposition of spermatozoa head pendular movements around the axis of forward motility and the other one around the head axis. This new type of tracks allows a simple quantitative analysis of hyperactivation of bull spermatozoa, first described in this report. The method is applied to investigations on efficacy of capacitation media and provides evidence for high individual differences of semen donors in capacitation success.

Animals↗
PubMed1993

Noradrenergic innervation of the hypothalamus of rhesus monkeys: distribution of dopamine-beta-hydroxylase immunoreactive fibers and quantitative analysis of varicosities in the paraventricular nucleus.

The distribution of noradrenergic processes within the hypothalamus of rhesus monkeys (Macaca mulatta) was examined by immunohistochemistry with an antibody against dopamine-beta-hydroxylase. The results revealed that the pattern of dopamine-beta-hydroxylase immunoreactivity varied systematically throughout the rhesus monkey hypothalamus. Extremely high densities of dopamine-beta-hydroxylase-immunoreactive processes were observed in the paraventricular and supraoptic nuclei, while relatively lower levels were found in the arcuate and dorsomedial nuclei and in the medial preoptic, perifornical, and suprachiasmatic areas. Moderate levels of dopamine-beta-hydroxylase immunoreactivity were found throughout the lateral hypothalamic area and in the internal lamina of the median eminence. Very few immunoreactive processes were found in the ventromedial nucleus or in the mammillary complex. Other midline diencephalic structures were found to have high densities of dopamine-beta-hydroxylase immunoreactivity, including the paraventricular nucleus of the thalamus and a discrete subregion of nucleus reuniens, the magnocellular subfascicular nucleus. A moderate density of dopamine-beta-hydroxylase immunoreactive processes were found in the rhomboid nucleus and zona incerta whereas little dopamine-beta-hydroxylase immunoreactivity was found in the fields of Forel, nucleus reuniens, or subthalamic nucleus. The differential distribution of dopamine-beta-hydroxylase-immunoreactive processes may reflect a potential role of norepinephrine as a regulator of a variety of functions associated with the nuclei that are most heavily innervated, e.g., neuroendocrine release from the paraventricular and supraoptic nuclei, and gonadotropin release from the medial preoptic area and mediobasal hypothalamus. Additionally, quantitative analysis of dopamine-beta-hydroxylase-immunoreactive varicosities was performed on a laser scanning microscope in both magnocellular and parvicellular regions of the paraventricular nucleus of the hypothalamus. The methodology employed in this study allowed for the high resolution of immunoreactive profiles through the volume of tissue being analyzed, and was more accurate than conventional light microscopy in terms of varicosity quantification. Quantitatively, a significant difference in the density of dopamine-beta-hydroxylase-immunoreactive varicosities was found between magnocellular and parvicellular regions, suggesting that parvicellular neurons received a denser noradrenergic input. These differential patterns may reflect an important functional role for norepinephrine in the regulation of anterior pituitary secretion through the hypothalamic-pituitary-adrenal stress axis.

Animals↗
PubMed1982

Cerebellar Purkinje cells are descended from a small number of progenitors committed during early development: quantitative analysis of lurcher chimeric mice.

Previous analysis of lurcher in equilibrium wild type aggregation chimeras revealed that the degeneration of cerebellar Purkinje cells (PCs) in lurcher mutants is due to a defect intrinsic to the PCs themselves. Thus, all of the PCs remaining in lurcher chimeric mice are descended from only the wild type embryo. In this study, we have determined the number of PCs in four lurcher chimeras and three wild type mice. The low number of wild type PCs (10,200) in half of the cerebellum of one chimera (chi 11) suggested that these cells might be descended from a single progenitor. This idea is strongly supported by the quantitative analysis of the PCs in the other animals. These numbers are integral multiples of the number of PCs in chi 11. We believe that each stepwise increase in the number of PCs in the chimeras is due to the addition of a single wild type cell to the progenitor pool. The existence of integral multiples implies that no other cells can contribute to the PC population after the progenitors become committed to forming PCs. We cannot know, however, whether the PC progenitors also give rise to other cell populations. The values of the integrals in wild type mice indicate that the entire PC population descends from a small number of progenitors (8 in C3H/HeJ mice). We calculate that these progenitors are committed during the neural plate to neural fold stage of development. Thus, the fate of the progenitors of the cerebellar PCs is restricted very early in neural development.

Aging↗
PubMed2004

Quantitative analysis of [Dmt(1)]DALDA in ovine plasma by capillary liquid chromatography-nanospray ion-trap mass spectrometry.

The synthetic opioid peptide analog Dmt-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA; [Dmt= 2',6'-dimethyltyrosine) is a highly potent and selective mu opioid-receptor agonist. A very sensitive and robust capillary liquid chromatography/nanospray ion-trap (IT) mass spectrometry method has been developed to quantify [Dmt(1)]DALDA in ovine plasma, using deuterated [Dmt(1)]DALDA as the internal standard. The standard MS/MS spectra of d(0)- and d(5)-[Dmt(1)]DALDA were obtained, and the collision energy was experimentally optimized to 25%. The product ion [ M + 2H-NH(3)](2+) (m/z 312.2) was used to identify and to quantify the synthetic opioid peptide analog in ovine plasma samples. The MS/MS detection sensitivity for [Dmt(1)]DALDA was 625 amol. A calibration curve was constructed, and quantitative analysis was performed on a series of ovine plasma samples.

Animals↗
PubMed1999

Methods for simultaneous quantitative analysis of eosinophil and neutrophil adhesion and transmigration.

Granulocytes play important roles in various inflammatory responses. The aim of this study was to develop in vitro methods to enable simultaneous analysis of eosinophil and neutrophil adhesion and transmigration in mixed granulocyte preparations. We used fibronectin-coated plates, with or without semipermeable inserts, to measure adhesion and transmigration. Granulocytes, from healthy blood donors, were stimulated with either interleukin (IL)-5 and eotaxin or N-formyl-methionyl-leucyl-phenylalanine (fMLP), during incubation in the wells. Three different detergents: n-octyl-beta-D-glucopyranoside (OG), Triton-X-100 or N-cetyl-N,N, N-trimethylammonium bromide (CTAB), were tested for their ability to cause lysis of granulocytes with minimal effect on eosinophil cationic protein (ECP) and myeloperoxidase (MPO) antigenicity. These two proteins were used for quantitative analysis of eosinophil and neutrophil adhesion/transmigration, and CTAB was the most efficient lysing reagent. Cell-recovery rates, based on ECP/MPO measurements, were > 95% in both assays. The adhesion and transmigration of eosinophils increased in a time-dependent manner upon stimulation with IL-5 and eotaxin. Eosinophil adhesion reached a plateau at 90 min of incubation and transmigration at 240 min. Neutrophils displayed a similar pattern of adhesion and transmigration upon activation with fMLP, reaching respective plateaux at 30 and 90 min. Our study shows that CTAB is an effective detergent for lysing granulocytes, yielding high and reproducible recovery rates of ECP and MPO. Measurement of ECP and MPO, as markers for cell counts, can therefore be used to quantify adhesive and transmigration properties of eosinophils and neutrophils in mixed granulocyte populations.

Blood Proteins↗
PubMed1999

Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis.

Direct immunogold labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a "control" tissue to identify preparation and labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during labeling, and several factors limiting quantitative analysis.

Animals↗
PubMed1985

Quantitative analysis of the topographical organization of olivocerebellar projections in the rat.

The inferior olivary projection to anterior and posterior regions of the cerebellar vermis was studied in the rat using the retrograde fluorescent double-labelling technique in combination with a computerized data collection system. A technique for quantifying and statistically analysing the point-to-point (or nucleotopic) organization of the projection is presented. It was found that one group of neurones in the medial accessory olivary nucleus projected to cerebellar lobules 6 or 7, and second group projected to lobules 4 or 5, while a third sent collaterals to both areas. In the rostral part of the nucleus these three groups were spatially separated (P less than 0.05), whereas caudally they were intermingled. It is concluded that a double-labelling method has distinct advantages over a single-tracer technique (such as horseradish peroxidase) in the investigation of the topographic organization of neuronal projections. Quantitative analysis is used here to overcome the problems presented by collateralization, inter-animal variation and the accurate representation of results from a series of animals.

Animals↗
PubMed1993

Detection and quantitative analysis of joint activity inflammation with 99Tcm-polyclonal human immunoglobulin G.

99Tcm-polyclonal human immunoglobulin G (HIG) scintigraphy was used to detect active joint inflammation and to obtain ratios of joint uptake in noninvolved and inflammatory joints. Imaging was performed at 4 and 24 h in 16 patients with rheumatoid arthritis (RA) and 16 with degenerative joint disease (control group). All joints (total of 1344) were scored for pain, swelling and visual analysis of uptake in both scans. Joint to background (J:B) ratios were also calculated. Clinical and visual scores correlated in both scans (r = 0.7, P < 0.01). In RA patients, 246 joints were clinically involved. Visual analysis of scans detected 213 (87%) of them at 4 h and 196 (80%) at 24 h. Joints with no pain or swelling showed significantly higher J:B ratios than the control group and lower ratios than joints clinically involved. In the control group, statistically significant differences in J:B ratios between the various joints were found, so it was necessary to establish a normal range for every joint. J:B ratios were significantly higher at 4 h than at 24 h in both groups of patients. 99Tcm-HIG scintigraphy allows detection and measurement of joint inflammation. Scans performed at 4 h are preferable to scans at 24 h. Quantitative analysis can measure more objectively the degree of activity and could be useful in the management of these patients.

Aged↗
PubMed1994

A quantitative analysis of antigen-presenting cell function: activated B cells stimulate naive CD4 T cells but are inferior to dendritic cells in providing costimulation.

Ligation of CD28 on CD4 Th1 clones and freshly isolated mixtures of naive and memory CD4 T cells triggered their T cell receptors (TCR) is sufficient to induce the costimulatory signals necessary for interleukin 2 (IL-2) production by these cells. CTLA-4-reactive ligands expressed on antigen-presenting cells (APC) are critical in providing costimulatory signals to these T cell populations. We demonstrate that these activation characteristics apply equally to purified naive CD4 T cells. Because B cell blasts express CTLA-4-reactive ligands and high levels of adhesion and major histocompatibility complex class II molecules, they would be expected to engage both the TCR and CD28 and consequently stimulate IL-2 production by naive CD4 T cells. Using purified populations of cells in limiting dilution cultures, we have carried out a quantitative analysis of the interaction between naive CD4 T cells and either activated B or dendritic cells. We demonstrate that B cell blasts stimulate a high frequency of naive CD4 T cells. Slight differences in TCR signaling efficiency between the two APC types were observed. Even at optimal peptide concentrations, however, the amount of IL-2 made by individual T cells was fourfold lower in response to B cell blasts than to dendritic cells. This relative deficiency of activated B cells was due to their inability to optimally costimulate naive CD4 T cells.

Abatacept↗
PubMed1986

Freeze-fracture study of the large myelinated club ending synapse on the goldfish Mauthner cell: special reference to the quantitative analysis of gap junctions.

The large myelinated club endings (LMCEs) of primary eighth nerve afferents form mixed synapses on the lateral dendrite of the giant Mauthner cell. The double replica freeze-fracture technique was employed to examine the intramembrane fine structure of these LMCE synapses. Morphological correlates of both chemical and electrical transmission were found at the LMCE synapses. Electrical synaptic junctions, or gap junctions, were located over much (10-20%) of the synaptic contact. These were seen in both pre-and postsynaptic membrane as tightly packed P face particle aggregates and corresponding aggregates of E face pits. Specializations characteristic of chemical synaptic junctions were most prominent at the periphery of the synaptic contact. These specializations consisted of postsynaptic E face particle aggregates which were subjacent to presynaptic active zones. The active zones were distinguishable as regions with an increased density of large particles and vesicle attachment sites represented by P face depressions and E face protuberances. Quantitative analysis of gap junction particle (connexon) number at five LMCEs revealed 24,000-106,000 connexons per LMCE. Comparison with data from electrophysiological studies of single LMCEs indicates that only a small fraction of the connexon channels are open at any given time during electrotonic transmission at an LMCE synapse.

Animals↗
PubMed2001

Quantitative analysis of phospholipid peroxidation and antioxidant protection in live human epidermal keratinocytes.

To characterize oxidative stress in phospholipids of normal human epidermal keratinocytes we metabolically labeled their membrane phospholipids with a natural oxidation-sensitive fluorescent fatty acid, cis-parinaric acid, and exposed the cells to two different sources of oxidants--a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethyl-valeronitrile), AMVN, and a superoxide generator, xanthine oxidase/xanthine. We demonstrated that both oxidants induced pronounced oxidation of four major classes of cis-parinaric acid-labeled phospholipids-phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol-in normal human epidermal keratinocytes that was not detectable as any significant change of their phospholipid composition. Vitamin E was effective in protecting the cells against phospholipid peroxidation. Since viability of normal human epidermal keratinocytes was not changed either by labeling or exposure to oxidants the labeling protocol and oxidative stress employed are compatible with the quantitative analysis of phospholipid peroxidation in viable cells.

Adult↗
PubMed2006

Computed tomography-based attenuation correction in neurological positron emission tomography: evaluation of the effect of the X-ray tube voltage on quantitative analysis.

BACKGROUND: The advent of dual-modality positron emission tomography/computed tomography (PET/CT) imaging has revolutionized the practice of clinical oncology by improving lesion localization and facilitating treatment planning for radiation therapy. In addition, the use of CT images for CT-based attenuation correction (CTAC) allows the overall scanning time to be decreased and a noise-free attenuation map (micromap) to be created. The most common procedure requires a piecewise linear calibration curve acquired under standard imaging conditions to convert the patient's CT image from low effective CT energy into an attenuation map at 511 keV. AIM: To evaluate the effect of the tube voltage on the accuracy of CTAC. METHODS: As different tube voltages are employed in current PET/CT scanning protocols, depending on the size of the patient and the region under study, the impact of using a single calibration curve on the accuracy of CTAC for images acquired at different tube voltages was investigated through quantitative analysis of the created micromaps, generated attenuation correction factors and reconstructed neurological PET data using anthropomorphic experimental phantom and clinical studies. RESULTS: For CT images acquired at 80 and 140 kVp, average relative differences of -2.9% and 0.7%, respectively, from the images acquired at 120 kVp were observed for the absolute activity concentrations in five regions of the anthropomorphic striatal phantom when CT images were converted using a single calibration curve derived at 120 kVp. Likewise, average relative differences of 1.9% and -0.6% were observed when CT images were acquired at 120 kVp and CTAC used calibration curves derived at 80 and 140 kVp, respectively. CONCLUSION: The use of a single calibration curve acquired under standard imaging conditions does not affect, to a visible or measurable extent, neurological PET images reconstructed using CTAC when CT images are acquired in different conditions.

Algorithms↗
PubMed1988

[Quantitative analysis of ventricular volumes by digital two-dimensional echocardiography].

The purpose of this study was to assess the usefulness of digital enhancement of two-dimensional echocardiographic images by cardiac cycle-synchronized summation and digital manipulation of the resulting images. We developed a system which provides for: (1) real-time storage of end-diastolic and end-systolic cardiac cycle images irrespective of rhythm variations; (2) exclusion from summation of end-diastolic and end-systolic images altered by respiratory movements or transducer displacements; (3) performance on the resulting images of various mathematical operations (3*3 convolutions, colour scale-guided manipulation of grey shades); (4) performance of complex mathematical calculations from the extracted edges including, in particular, reconstruction of ventricular volumes by Simpson's crossed method from a transverse and an apical section or from two apical sections. This method was used to collect echocardiographic images from 32 patients who had undergone left ventricular quantitative angiography during the same week. Qualitatively, digital enhancement of two-dimensional echocardiographic images undoubtedly facilitated the identification of endocardial edges and reduced image noise, notably in patients whose ventricular edges were virtually unrecognizable by any other method. Moreover, summation improved correlations with angiography and reduced the variability of quantitative ventricular volume assessment. Finally, the ease and rapidity with which this "real-time" system can be used enables quantitative analysis of left ventricular function to be routinely carried out.

Adult↗
PubMed1990

Immunohistochemical detection of major histocompatibility complex antigens and quantitative analysis of tumour-infiltrating mononuclear cells in renal cell cancer.

In order to investigate the anti-tumour immune responsiveness of patients with renal cell cancer (RCC), we examined 30 such patients for the degree of expression of major histocompatibility complex (MHC) class I and class II antigens on RCC and the populations of tumour-infiltrating mononuclear cells (TIM). Normal renal tubular cells expressed class I but not class II antigens. Most of the tumour cells expressed class I antigens in 25 (83%) cases, but the proportion of such cells was reduced in five cases, three of which were of granular cell type histologically. Class II antigens were detected in all specimens with class I positivity. Various numbers of TIM were detected in 25 cases, being composed mainly of T cells and a smaller number of macrophages. Examination for the phenotype of T cells showed that CD8-positive cells were the dominant population. B cells were not detected. Quantitative analysis revealed that the numbers of TIM were significantly lower in cases showing class I reduction than in those with normal class I expression. Therefore, it was clear that class I antigens were preserved in RCC cells in most cases. Furthermore, a higher rate of reduction of class I antigens was observed in cases of granular cell type, which has been reported to have a worse prognosis than the clear cell type. The present data suggest that degree of the expression of MHC class I antigen on RCC might influence the host immune responsiveness against it.

Adult↗
PubMed1987

Quantitative analysis of myocardial infarction in (NZW x BXSB)F1 hybrid mice with systemic lupus erythematosus and small coronary artery disease.

Male (NZW x BXSB)F1 mice ([W x B]F1) were used as a model for small coronary artery disease. The mortality rate for 162 mice was 0% at 12 weeks and 37% at 24 weeks. The incidence of myocardial infarction (MI) was 0% at 12 weeks, 22% at 16 weeks, 39% at 20 weeks, and 53% at 24 weeks. In 29 of the 35 (W x B)F1 male mice with MI, small multiple infarctions were noted in the right ventricular free wall and anterior, lateral, posterior, and septal ventricular walls. In 25 of the 29 hearts with multiple infarcts, the infarcts in the same heart were at the same histologic stage. Twenty-one of these hearts showed only replacement fibrosis, and 4 hearts showed only granulation. The remaining 4 hearts with multiple infarcts exhibited coagulation necrosis plus fibrosis or granulation. Quantitative analysis of the infarct size revealed that in the 35 (W x B)F1 males with MI, the relative area of MI (%MI) was significantly larger in the right ventricular free wall (6.7% +/- 8.4%) than in the ventricular septum (1.9% +/- 2.4%) or in the left ventricular free wall (2.1% +/- 2.5%). The %MI was greatest in the right third (3.6% +/- 5.4%) of the ventricular septum and in the outer third (2.9% +/- 3.3%) of the left ventricular free wall. The %MI did not increase with age. In 11 of the 35 (W x B)F1 mice, 27 intramural small arteries showed marked obliterative lesions. Most of them were in the right ventricular free wall, the right third of the ventricular septum, or the outer third of the left ventricular free wall. There was no evidence of stenosis in the extracardiac major coronary arteries. In addition, the infarct showed a whirlpool-like configuration, and x-ray photographs revealed that the small intramural coronary arteries had a whirlpool-like configuration. It is concluded that in (W x B)F1 males multiple small infarcts appear at the same time due to small coronary artery disease. The whirlpool configuration of the infarct reflects the special anatomy of the intramural coronary arteries in the mice.

Animals↗
PubMed1993

A light and electron microscopic quantitative analysis of the innervation of axillary lymph nodes in juvenile and old rats.

The innervation of axillary lymph nodes from 20 juvenile rats (aged < 6 wk) was compared with that of 20 old rats (aged > 2 y). One half of each group was investigated by light microscopy on silver-impregnated paraffin sections, the other half by electron microscopy. The lymph nodes of the old animals were larger than those of juvenile animals, as derived from the volumes analysed by light microscopy. By both light and electron microscopy, the nerves were found to be confined almost exclusively to the medulla and were frequently associated with groups of plasma cells. Nerves identified as cortical in location were consistently found only in cortical areas adjacent to the medulla. In the old animals the nerves were thicker and more prominent in the light microscopic preparations. No such differences were noted at the ultrastructural level. Myelinated nerves were found in all lymph nodes, except in a single old animal. Quantitative analysis of the nerves revealed a significant increase in the density of innervation of the medulla in the old animals both by light and electron microscopy. By light microscopy there also appeared to be a significant increase in the innervation density of the cortex, but these nerves were considered to be mislocalized because of difficulty in precise localisation at this level of magnification. On light microscopy, a significant increase in the incidence of nerve branching was found in the nodes of the old animals.

Aging↗
PubMedDate not supplied

Isolation and HPLC quantitative analysis of antioxidant flavonoids from Alternanthera tenella Colla.

Phytochemical analysis of the antioxidant ethanolic extract of Alternanthera tenella Colla led to the isolation of six flavonoids, acacetin 8-C-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranoside] (1), 2"-O-alpha-L-rhamnopyranosyl-vitexin (2), 2"-O-beta-D-glucopyranosyl-vitexin (3), vitexin (4), quercetin (5) and kaempferol (6). All the structures were established by ESI-MS and NMR spectroscopic methods. Antioxidant capacity of extract, fractions and isolated compounds was determined using the oxygen radical absorbance capacity (ORAC) assay and extract, fractions and flavonoids isolated showed antioxidant activity in vitro. Moreover, the total soluble phenolic contents of the extract and fractions were measured using the Folin-Ciocalteau reagent and the quantitative analysis of flavone C-glycosides major constituents was performed by HPLC.

Antioxidants↗
PubMed2004

Quantitative analysis of microtubule transport in growing nerve processes.

In neurons, tubulin is synthesized primarily in the cell body, whereas the molecular machinery for neurite extension and elaboration of microtubule (MT) array is localized to the growth cone region. This unique functional and biochemical compartmentalization of neuronal cells requires transport mechanisms for the delivery of newly synthesized tubulin and other cytoplasmic components from the cell body to the growing axon. According to the polymer transport model, tubulin is transported along the axon as a polymer. Because the majority of axonal MTs are stationary at any given moment, it has been assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent "stop and go" MT transport has been difficult to detect or to exclude by using direct video microscopy methods. In this study, we measured the translocation of MT plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required for delivery of newly synthesized tubulin to the growing nerve processes.

Animals↗