A cytochemical study of pituitary glands of 1- to 15-day-old rats utilizing the aldehyde-fuchsin staining technique.
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Aldehydes↗
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Differential collagen stain by an acid fuchsin, iron, flavianic acid mixture. A note on ferrous sulfate hematoxylin.
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Animals↗
Subdivision of the mitotic cycle into eleven stages, on the basis of the chromosomal changes observed in mouse duodenal crypt cells stained by the DNA-specific Feulgen reaction.
The Feulgen reaction has been utilized to localize DNA in nuclei throughout the cycle of mouse duodenal crypt cells using Epon-embedded 1 micron thick sections. The observed changes indicate that the 12.3 h long mitotic cycle of these cells can be subdivided into eleven stages, seven of which take place during the interphase. Computer measurements of Feulgen-stained nuclei and previous radioautographic studies indicate that DNA synthesis begins during stage I and ends during stage IV. The staining pattern shows no distinctive feature in the nuclei of the 1.5 h long stage I. Thereafter, marked changes occur during the rest of the interphase--that is during the 6.3 h that precede karyokinesis and the 3.5 h that follow it. Thus, at stage II the background of the nuclei darkens; at stage III, there appear stained threads interpreted as densifying chromosomes and dots interpreted as chromomeres, both of which thicken from 0.2 to 0.4 micron; at stage IV they further thicken to about 0.5 micron and at stage V, to about 0.7 micron. At this stage, which approximately corresponds to prophase, the intensely stained, discrete dots are localized within the less intensely stained sausage-shaped threads. As the breakup of the nuclear envelope introduces stage VI, whose early part corresponds to prometaphase, the intensely stained dots become close to one another within the threads and eventually fuse. The staining of the threads thus intensifies, and, by the late part of the stage that corresponds to metaphase, they have become the homogeneously dense metaphase chromosomes. At stage VII, the anaphase chromosomes reach each pole where they associate into a compact mass.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Morphology and DNA degeneration during autoschizic cell death in bladder carcinoma T24 cells induced by ascorbate and menadione treatment.
Feulgen and actin-phalloidin staining as well as gel electrophoresis have been employed in conjunction with cell ultrastructure to describe the effects of 1-, 2-, and 4-hr ascorbate (VC), menadione (VK(3)), and ascorbate:menadione (VC:VK(3)) treatments on the T24 human bladder carcinoma cell line. T24 cells exposed to VC alone display blebs and other superficial membrane defects related to membrane alterations and to superficial cytoskeleton changes. VK(3) treatment damages the cell nucleus and organelles, leads to the redistribution of the organelles in the perikaryon as a consequence of cytoskeletal damage, and results in cytoplasmic self-excisions. After VC:VK(3) treatment, the cells show exaggerated alterations characteristic of each vitamin treatment alone, including damaged mitochondria, self-excision of organelle-free pieces of cytoplasm, and extrusion of the perikaryon containing a nucleus surrounded by the damaged organelles. The nuclear envelope appears intact and contains chromatin that decondenses and dissipates. During the cellular demise that concludes with apparent karyolysis, the cells significantly decrease their size and alter their shape. However, the cisterns of rough endoplasmic reticulum are undamaged, but may become dilated. Since the cellular phenomena leading to cell death differ morphologically from apoptosis and necrosis, but entail self-cutting without nuclear bodies, this new form of cell death was called autoschizis. In addition, gel electrophoresis and Feulgen staining demonstrate that autoschizis is accompanied by random DNA degeneration.
Ascorbic Acid↗
Feulgen reaction study of novel threadlike structures (Bonghan ducts) on the surfaces of mammalian organs.
Threadlike structures on the surfaces of internal organs, which are thought to be part of the Bonghan duct system, were first reported about 40 years ago, but have been largely ignored since then. Recently, they were rediscovered, and in this study we discuss the Feulgen reaction that specifically stains DNA in order to identify these structures on the surface of rabbit livers as part of the Bonghan system. The distribution, shapes, and sizes of their nuclei are found to be similar to those of intravascular threadlike structures. The endothelial nuclei are rod-shaped, 10-20 mum long, and aligned in a broken-line striped fashion. The threadlike structure consists of a bundle of several subducts, which is a characteristic feature of Bonghan ducts and distinguishes them morphologically from lymphatic vessels. In addition, the Feulgen reaction clearly demonstrates that the subducts pass through a corpuscle, which is usually irregular or oval-shaped and is connected to two or several threadlike structures that form a web on the surfaces of organs. Furthermore, spherical granules of about 1 mum in diameter are detected in the subducts. These granules were well stained by using the Feulgen reaction, which implies that they contain DNA. According to previous reports, a granule is a type of microcell and plays an essential role in the physiology and therapeutic effect of the Bonghan system and acupuncture. This role has yet to be elucidated.
Acupuncture Points↗
Malachite green-conjugated microtubules as mobile bioprobes selective for malachite green aptamers with capturing/releasing ability.
We have developed a novel mobile bioprobe using a conjugate of a kinesin-driven microtubule (MT) and malachite green (MG) as a platform for capturing MG RNA aptamers. The fluorescence of MG increases when it is bound to an MG aptamer, allowing MT-MG conjugates to work as sensors of RNA transcripts containing the MG aptamer sequence. Kinesin motor proteins provide an effective driving force to create mobile bioprobes without any manipulation. Although the fluorescence of a small number of MG-binding aptamers is low, the self-organization of tubulins into MTs enables the microscopic observation of the bound aptamers by collecting them on MTs. We demonstrate that MT-MG conjugates can select target aptamers from a transcription mixture and transport them without losing their inherent motility. Because the MG aptamer binds MG in a reversible manner, MT-MG conjugates can conditionally load and unload the target aptamers. This is one advantage of this system over the molecular probes developed previously in which reversible unloading is impossible due to high-affinity binding, such as between avidin and biotin. Furthermore, an MT-MG conjugate can be used as a platform for other MG aptameric sensors with recognition regions for various target analytes optimized by further selection procedures. This is the first step to applying living systems to in vitro devices. This technique could provide a new paradigm of mobile bioprobes establishing high-throughput in vitro selection systems using microfluidic devices operating in parallel.
Aptamers, Nucleotide↗
Sentinel node detection after preoperative short-course radiotherapy in rectal carcinoma is not reliable.
BACKGROUND: Sentinel node (SN) detection may be used in patients with colonic carcinoma. However, its use in patients with rectal carcinoma may be unreliable. To address this, SN detection was evaluated in patients with rectal carcinoma after short-course preoperative radiotherapy. METHODS: Patent Blue V (1-2 ml) was injected peritumorally and submucosally directly after total mesorectal excision (TME) in 34 patients. The first one to four blue lymph nodes were categorized as SNs. All lymph nodes (non-SNs and SNs) were examined by conventional haematoxylin and eosin stained sections. If the SN was negative for metastasis, additional sections were immunostained with anticytokeratin CK7/8. In addition, SN detection was performed in 57 patients with colonic carcinoma. RESULTS: A SN was identified in 26 of 34 patients with rectal carcinoma. In three the SN was the only positive lymph node. There were six false-negative SNs (sensitivity 40 per cent) and two patients were upstaged. By contrast, SN detection was possible in 56 of 57 patient with colonic carcinoma with a sensitivity of 90 per cent, and four patients were upstaged. CONCLUSION: The SN procedure for rectal carcinoma is not reliable in combination with TME and preoperative short-course radiotherapy.
Adult↗
Scanning cytophotometric analysis of myocardial nucleic acid and chromatin changes in soman toxicated rabbits.
Myocardial nucleic acid responses were analysed in New Zealand White rabbits 20 min-1 h and 6-8 h following single subcutaneous injections of soman (20, 30, or 40 micrograms kg-1). Scanning-integrating microdensitometry was used to quantify Azure B-RNA and Feulgen-DNA (F-DNA) levels, and changes in the susceptibility of chromatin to Feulgen acid hydrolysis (F-DNA reactivity) of individual ventricular myocardial cells. With a dosage of 20 micrograms kg-1 soman, no RNA alterations were evidenced at 1 h whereas at 6-8 h myocardial cells exhibited higher RNA levels and an increase in F-DNA reactivity of chromatin. With dosages of 30 and 40 micrograms kg-1 soman there was an augmentation in RNA levels and in the acid hydrolysability of nuclear chromatin at both 20 min-1 h and 6-8 h. It is postulated that the observed cellular transformations represent a compensatory augmentation in myocardial metabolic functioning presumably in response to an increased functional demand on the ventricular myocardium. The absence of cytopathic or cytochemical evidence of impairment in nucleic acid metabolism is inconsistent with the premise that soman exerts direct cytotoxic effects on rabbit myocardium.
Animals↗
Cytophotometric assessment of Feulgen-DNA and coomassie-total cell protein in adrenocortical fasciculata cells of noise-exposed Wistar rats.
The present investigation was undertaken to examine possible noise-induced alterations in adrenal fasciculata cell (AFC) metabolism, and also to determine if the magnitude of these changes differs in male versus female rats. Wistar rats approximately 3 months old were exposed to intense noise for 60 min (100 dB, re 2 x 10(-5) N(m2)-1, 350-20,000 Hz); control rats were housed under identical conditions, at an ambient noise level of 40-60 dB. Adrenal fasciculata cells (AFC) from each animal were examined for noise-induced alterations in Feulgen-DNA reactivity (as an indicator of chromatin template activity) and Coomassie-total cell protein levels using scanning-integrating cytophotometry. The results provide evidence that intense noise elicited a marked AFC metabolic enhancement in both male and female rats; the degree of this enhancement was more pronounced in males. This disparity may be due to pre-existing differences in male versus female AFC enzymatic capability and subsequent responsiveness to noise-induced activation.
Animals↗
Microdensitometric measures of cytoplasmic RNA and total protein in pyramidal neurons of the insular cortex and midfrontal gyrus in patients with Alzheimer's disease.
Scanning and integrating microdensitometry of azure B- and Coomassie brilliant blue G-stained tissue sections was used to measure the levels of RNA and protein, respectively, in pyramidal neurons of the insular cortex (INS) and midfrontal gyrus (MFG) in patients with Alzheimer's disease (AD) and age-matched, nondemented control subjects. AD was associated with a decreased neuronal RNA (by 7.4 per cent) and protein (by 28.7 per cent) content in INS. Although the neuronal RNA content was maintained at the control amounts in MFG, the average protein level was lower (14.7 per cent) in AD patients. These results demonstrate a disease-related impairment in metabolic function in two brain regions connected via discrete corticocortical pathways. Such findings support the hypothesis that a primary site of pathology occurs in AD, and specific neural deficits occur secondarily in certain connected brain regions.
Aged↗
Aptamer to ribozyme: the intrinsic catalytic potential of a small RNA.
The discovery of RNA-based catalysis 23 years ago dramatically changed the way biologists and biochemists thought of RNA. In the recent past, several ribozymes structures have provided some answers as to how catalysis is accomplished and how it relates to RNA structure and folding. However, there is still little information as to how catalytic activity evolved. Here we show that the small malachite green-binding aptamer has intrinsic catalytic potential that can be realized by designing the proper substrate. The charge distribution within the RNA binding pocket stabilizes the transition state of an ester hydrolysis reaction and thus accelerates the overall reaction. The results suggest that electrostatic forces can contribute significantly to RNA-based catalysis. Moreover, even simple RNA structures that have not been selected for catalytic properties can have a basic catalytic potential if they encounter the right substrate. This provides a possible starting point for the molecular evolution of more complex ribozymes.
Aptamers, Nucleotide↗
3-hydroxybenzene 1,2,4-trisphosphate, a novel second messenger mimic and unusual substrate for type-I myo-inositol 1,4,5-trisphosphate 5-phosphatase: Synthesis and physicochemistry.
3-Hydroxybenzene 1,2,4-trisphosphate 4 is a new myo-inositol 1,4,5-trisphosphate analogue based on the core structure of benzene 1,2,4-trisphosphate 2 with an additional hydroxyl group at position-3, and is the first noninositol based compound to be a substrate for inositol 1,4,5-trisphosphate 5-phosphatase. In physicochemical studies on 2, when three equivalents of protons were added, the (31)P NMR spectrum displayed monophasic behaviour in which phosphate-1 and phosphate-2 behaved independently in most of the studied pH range. For compound 4, phosphate-2 and phosphate-4 interacted with the 3-OH group, which does not titrate at physiological pH, displaying complex biphasic behaviour which demonstrated co-operativity between these groups. Phosphate-1 and phosphate-2 strongly interacted with each other and phosphate-4 experienced repulsion because of the interaction of the 3-OH group. Benzene 1,2,4-trisphosphate 2 is resistant to inositol 1,4,5-trisphosphate type I 5-phosphatase catalysed dephosphorylation. However, surprisingly, 3-hydroxybenzene 1,2,4-trisphosphate 4 was dephosphorylated by this 5-phosphatase to give the symmetrical 2,3-dihydroxybenzene 1,4-bisphosphate 16. The extra hydroxyl group is shown to form a hydrogen bond with the vicinal phosphate groups at -15 degrees C, and (1)H NMR titration of the ring and hydroxyl protons in 4 shows the OH proton to be strongly stabilized as soon as the phosphate groups are deprotonated. The effect of the phenolic 3-OH group in compound 4 confirms a critical role for the 6-OH group of the natural messenger in the dephosphorylation mechanism that persists even in radically modified analogues.
Chemical Phenomena↗
Laparoscopic sentinel lymph node procedure using a combination of patent blue and radioisotope in women with cervical carcinoma.
BACKGROUND: The authors evaluated the feasibility of a laparoscopic sentinel lymph node (SN) procedure with combined radioisotopic and patent blue labeling in patients with cervical carcinoma. METHODS: Thirteen women (median age, 52.5 years) with cervical carcinoma (Stage Ia2 in 1 patient, Stage Ib1 in 10 patients, Stage Ib2 in 1 patient, and Stage IIa in 1 patient) underwent a laparoscopic SN procedure using an endoscopic gamma probe after both radioactive isotope and patent blue injections. After the procedure, all patients underwent complete laparoscopic pelvic lymphadenectomy and either laparoscopic radical hysterectomy (eight patients) or the Schauta-Amreich operation (five patients). RESULTS: SNs (mean, 1.7 SNs per patient; range, 1-3 SNs per patient) were identified in 12 of 13 patients. A median of 10.5 pelvic lymph nodes per patient (range, 4-17 pelvic lymph nodes per patient) were removed. No lymph node involvement was detected in SNs with hematoxylin and eosin staining. Immunohistochemical studies identified four metastatic SNs in two patients, with micrometastases in two SNs from the first patient and isolated tumor cells in two SNs from the second patient. No false-negative SN results were obtained. CONCLUSIONS: The results of this study suggest that SN detection with a combination of radiocolloid and patent blue is feasible in patients with cervical carcinoma. The combination of laparoscopy and the SN procedure permitted minimally invasive management of early-stage disease.
Adult↗
Prospective evaluation of a novel approach for the use of a quantitative galactose oxidase-Schiff reaction in ductal fluid samples from women with breast carcinoma.
BACKGROUND: The galactose oxidase-Schiff reaction (GOS) yields positive findings in a number of malignant solid tumors. The goals of the current study were to develop a novel technique for quantifying GOS reactivity in nipple aspirate fluid (NAF) samples from women with invasive breast carcinoma and to assess the clinical utility of the technique in this setting. METHODS: Patients with biopsy-proven unilateral invasive breast carcinoma were eligible for study entry. Before definitive surgery, NAF samples were obtained from healthy breast tissue and malignant breast tissue from 23 women with breast carcinoma. Under blind conditions with respect to clinical data, 10 microL NAF samples were applied to a glass fiber membrane and incubated with 100 microL galactose oxidase and 1 mL Schiff reagent. The stain was developed and the color reaction quantitated by measuring hue (shade) and chroma (intensity) using a spectrophotometer. RESULTS: GOS reactivity was quantitated using two color parameters, hue and chroma. Because chroma varies with concentration, this measurement was adjusted for the concentration of NAF in each sample. After adjustment for NAF concentration, chroma was found to be statistically significantly different in the affected breast tissue sample and the healthy contralateral internal control sample (P = 0.001). CONCLUSIONS: A quantitative measure of GOS reactivity based on spectrophotometric measurement of intensity of color has been developed and was found to be significantly different in the affected breast compared with the unaffected breast in the current population of patients with breast carcinoma. The preliminary results support further exploration of this novel quantitative test in patients with breast carcinoma.
Adult↗
High resolution DNA content measurements of mammalian sperm.
The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +/- 7 degrees. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative DNA content of these two populations in sperm from normal mice and those with the Cattanach [7 to X] translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.
Acriflavine↗
The "central dogma" of flow cytometry.
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Coloring Agents↗
Cytochemical analysis of the chromatin of liver cell nuclei and nuclei of oval cells by means of the Feulgen hydrolysis curve in rats treated by thioacetamide.
The present study is an analysis of the Feulgen hydrolysis characteristics in nuclei of liver cells and oval cells in rats treated by thioacetamide (TAA) and of liver cells in control rats. The curves show a double-peaked pattern. A slower hydrolysis is noted in the first peak after TAA treatment. This suggests an alteration of the acid-labile part of the chromatin. The curve obtained in the oval cells is different from the one in the liver cells. The implications of these differences are discussed with respect to development of cholangiocarcinomas.
Acetamides↗
The G0 in equilibrium G1 transitions of human lymphocytes as monitored by quantitative 14C-uridine autoradiography and high-resolution image analysis.
Human lymphocytes, transferred in culture into medium conditioned with phytohemagglutinin, were studied with the combined use of quantitative 14C-uridine autoradiography and high-resolution image analysis. During the first 24 h before the onset of DNA synthesis it was possible to study pure G0 and G1 populations. The return of the proliferating cells into the G0 state was clearly monitored in an RNA synthesis rate/DNA content plot during 1-week growth. On days 3-6, the proliferation decreased progressively, and more cells accumulated in the quiescent G0 state. Geometric, densitometric, and textural parameters evaluated from the Feulgen-stained nuclei of the same cells indicated that the decondensation/condensation processes of the nuclear chromatin during the G0 in equilibrium G1 transitions paralleled the RNA synthesis activity. The results help to associate descriptors of nuclear morphology, as derived from image analysis, with functional parameters during activation and return to quiescence of human lymphocytes.
Autoradiography↗