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[Effect of macrophage-derived growth factor on the cell cycle kinetics of cultured arterial smooth muscle cells].

Effects of macrophage-derived growth factor on the progression of the cell cycle of cultured aortic smooth muscle cells were investigated by using rabbit peritoneal macrophage conditioned-medium (M phi-CM) as the source of the growth factors. DNA content of individual SMC in the cell population which had been exposed either to platelet-poor plasma serum (PPPS) or to M phi-CM was also measured by flow cytometry and microspectrophotometry, as well as by phase contrast microscopy. The results showed that the cells quiescent in PPPS medium had a high population not only in G0/G1 but also in G2 phase. This indicated that the cell cycle process was blocked both in G0/G1 and G2 phase. The cell cycle distribution of SMCs exposed to M phi-CM for 48 hours was very similar to that cultured in 10% fetal calf serum medium. It is suggested that M phi-CM can promote the cell growth-arrested in ppps medium passing through G0/G1 into S phase as well as through G2 into M phase.

Animals↗

[Study on DNA content and nucleus/cytoplasm ratio of esophageal epithelium during malignant change].

DNA content and nucleus/cytoplasm ratio were assayed in the paracancerous mucosa from 37 cases of esophageal carcinoma by microspectrophotometry (Leitz MPV3). The histopathological diagnosis was made at the same area. In normal epithelium and simple hyperplasia, the relative DNA content was within the range of 2C-4C; cells with DNA content of 4C or greater than 4C were found mostly in invasive carcinoma. There were two different types of DNA content histogram observed in atypical hyperplasia and carcinoma in situ. One type was similar to simple hyperplasia and the other type was similar to invasive carcinoma. These results indicated that two different malignant features might be obtained under the same histopathological diagnosis. The nucleus/cytoplasm ratio in simple hyperplasia was close to that of normal mucosa. In atypical hyperplasia and carcinoma in situ, especially in the middle and superficial layers of the epithelium, the nucleus/cytoplasm ratio increased markedly, which indicated that the cells in these layers were poorly differentiated.

Cell Nucleus↗

Microspectrophotometric studies on single crystals of the tryptophan synthase alpha 2 beta 2 complex demonstrate formation of enzyme-substrate intermediates.

Microspectrophotometry of single crystals of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium is used to compare the catalytic and regulatory properties of the enzyme in the soluble and crystalline states. Polarized absorption spectra demonstrate that chromophoric intermediates are formed between pyridoxal phosphate at the active site of the beta subunit and added substrates, substrate analogs, and reaction intermediate analogs. Although the crystalline and soluble forms of the enzyme produce some of the same enzyme-substrate intermediates, including Schiff base and quinonoid intermediates, in some cases the equilibrium distribution of these intermediates differs in the two states of the enzyme. Ligands which bind to the active site of the alpha subunit alter the distribution of intermediates formed at the active site of the beta subunit in both the crystalline and soluble states. The three-dimensional structures of the tryptophan synthase alpha 2 beta 2 complex and of a derivative with indole-3-propanol phosphate bound at the active site of the alpha subunit have recently been reported (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 264, 17857-17871). Our present findings help to establish experimental conditions for selecting defined intermediates for future x-ray crystallographic analysis of the alpha 2 beta 2 complex with ligands bound at the active sites of both alpha and beta subunits. These crystallographic studies should explain how catalysis occurs at the active site of the beta subunit and how the binding of a ligand to one active site affects the binding of a ligand to the other active site which is 25 A away.

Crystallization↗

Coupled enzymatic reactions measured in a single protein crystal from myogen A.

Pairwise coupled reactions of fructose-1,6-bisphosphate aldolase and sn-glycerol-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and D-glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and sn-glycerol-3-phosphate dehydrogenase have been detected by microspectrophotometry in single crystals obtained from myogen A in the presence of polyethylene glycol. Microspectrophotometric measurements with polarized light demonstrate that the protein molecules are oriented and that NADH is bound with a definite orientation to the dehydrogenases within the crystal.

Animals↗

[Autoradiographic study on etorphine and phencyclidine specific binding sites in rabbit mesenteric artery].

Autoradiography was used to study the localization of kappa and PCP/sigma receptors in the blood vessels. Slices of rabbit mesenteric arteries were incubated with 0.4 nmol/L [3H]etorphine or/and 10 mumol/L etorphine for 45 min at 25 degrees C, or incubated with 5.2 nmol/L [3H]PCP or/and 20 mumol/L PCP for 60 min at 4 degrees C. Then, slices were covered with emulsion coated coverslip and kept for 8-10 wk at 4 degrees C. The results were as follows: Microscopy Autoradiographic analysis indicated that etorphine and PCP specific binding sites were both located in the outer-layer and the smooth muscle of the artery. However, most of these binding sites were lost in the 6-OHDA pretreated arteries in which the adrenergic nerve endings were destroyed. Microspectrophotometry The absorbance (A) of [3H]etorphine autoradiographic density for total binding (TB) and nonspecific binding (NSB) in control group were 0.416 +/- 0.056 and 0.044 +/- 0.011, respectively (P less than 0.01), and for TB after incubation with 6-OHDA was 0.068 +/- 0.013 which was different from the A value of TB in the control (P less than 0.01). The A value of [3H]PCP autoradiographic density for TB and NSB in the control were 0.546 +/- 0.087 and 0.023 +/- 0.060, respectively (P less than 0.01), and for TB after incubation with 6-OHDA was 0.065 +/- 0.015 which was significantly less than that of TB in the control group (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Microspectrophotometric determination of DNA in oral lesions.

The DNA content of the epithelial basal cells of oral lesions was determined by Feulgen microspectrophotometry. The DNA histograms of the radicular cysts showed a definite peak in the diploid range and a slight deviation to the right. The keratocysts also showed a well-defined modal peak in the diploid range; however, another cell population defined a peak in the tetraploid range. In leukoplakias of the oral cavity, two different profiles of DNA distribution were found. Where both the clinical and microscopic traits defined a nondysplastic lesion, the histogram showed a diploid peak and a percentage of cells in diploid-tetraploid range. For the more active lesion, a shift of the mode towards higher values was evident. The microspectrophotometric studies of oral epidermoid carcinomas showed a broad distribution towards the right and ill-defined modal values.

Carcinoma, Squamous Cell↗

Diagnosis of benign and malignant cervical specimens by multifiberoptic microspectrophometry and by flow cytometry.

Two techniques for the automation of mass screening for cervical cancer were studied. Microspectrophotometry was tried first, using a novel multifiberoptic scanning system that measured the nuclear size and DNA content of cells in routine smears restained by the Feulgen technique. Specimen diagnoses were based on the percentages of cell types present, as determined by thresholds set for the two parameters. While this method gave good results in the automated detection of severe dysplasias and carcinomas, with only 3 of 72 cases misdiagnosed as negative (4.2%), it had a 22.9% false-positive rate (misdiagnosing 24 of 105 "benign" cases) and a 30.3% false-negative rate for adenocarcinomas (10 of 33 cases misclassified). The second approach involved flow cytometric measurements of specimens that were double stained for the assessment of both the DNA and RNA content, with the results analyzed by preset windows in a two-dimensional plane. This technique gave a 6.1% false-negative rate in 49 positive specimens and a 32.3% false-positive rate in 102 benign specimens, with an overall correct classification rate of 76.2%, including adenocarcinomas.

Female↗

Megakaryocytopenia in W/Wv mice is accompanied by an increase in size within ploidy groups and acceleration of maturation.

Megakaryocytopoiesis was evaluated in W/Wv mice and their normal +/+ littermates to analyze the mechanisms by which normal platelet production is maintained in W/Wv mice even though numbers of megakaryocytes are low. Relative sizes of megakaryocytes, and their nuclei and cytoplasm, were measured microscopically in bone marrow smears, and the ploidy of the same cells was measured by two-wavelength microspectrophotometry. Maturation rate of megakaryocytes was estimated after they were labeled with tritiated thymidine. W/Wv megakaryocytes were macrocytic: average cell size was increased in each ploidy group. The increase in cytoplasmic area exceeded that of the nucleus. Further analysis of the predominant 16N ploidy group revealed that the increase in average cell size was due to depletion of cells of small size. Megakaryocytes matured more rapidly than normal in W/Wv mice. These results showed that megakaryocyte size and ploidy can be regulated separately. They suggest that alterations in cell growth and maturation may be mechanisms by which the organism can compensate for a deficiency in numbers of megakaryocytes, but they do not define the mechanism by which the deficiency may be sensed or by which the compensatory changes may be mediated. This is a US government work. There are no restrictions on its use.

Animals↗

[Inhibition of acetylcholinesterase at pupil-related central nuclei by organophosphorus pesticide (fenthion)--an experimental study].

An organophosphorus pesticide (OP) inhibits acetylcholinesterase (AChE) of various organs, especially in cholinergically innervated organs. Pupil constriction is a major sign of an ocular symptom in the patient with acute OP intoxication, but the etiology is unknown. The present experiments were intended to elucidate a central mechanism of pupil size in which AChE and OP are involved. Fenthion (dimethyl 4-methylthion-m-tolyl phosphorothionate) of 5mg/day body weight) was subcutaneously administered dorsally in Wistar rats for 14 days consecutively in the longest group. Histochemical staining and microspectrophotometry were used to analyze the intensity of the AChE activity in the iris, the pretectal nucleus and Edinger-Westphal (EW) nucleus on the 2nd, 4th, 6th, 10th and 14th day of administration. The pupil was measured after 20 minutes of dark adaptation. The pupil constricted on the 6th day and most remarkably on the 10th day. The AChE activity in the iris decreased remarkably on the 4th day. Miosis was not seen on the 4th day. The AChE activity in the pretectal nucleus decreased remarkably on the 6th day. The AChE activity in the EW nucleus reduced gradually from the 6th day and 62% reduction of initial value was seen on the 14th day. Intense miosis was seen on the 10th day. In conclusion inhibition of AChE activity was detected first in the iris, thereafter in the pretectal nucleus and the EW nucleus in close correlation with the miotic pupil. Therefore, miosis associated with OP intoxicated patients may parallel the central inhibition of AChE in pupil-related centers.

Acetylcholinesterase↗

[Determination of cell nuclear DNA content in leiomyoma and leiomyosarcoma of gastro-intestinal tract (G-I)].

Nuclear DNA content was measured by microspectrophotometry in 15 biopsy specimens from patients with G-I smooth muscle tumors (3 leiomyomas and 12 leiomyosarcomas subdivided into 3 groups with 4 cases to each). The mean DNA value increased steadily as follows: leiomyomas (14.39 +/- 0.62 Au); leiomyosarcoma Grade I (19.78 +/- 2.39 Au); Leiomyosarcoma Grade II (26.39 +/- 1.60 Au); leiomyosarcoma Grade III (30.66 +/- 2.39 Au). The difference of DNA value in the 4 groups had statistical significance (P less than 0.05-0.01). These results suggest that microspectrophotometric measurement of nuclear DNA content may serve as an objective quantitative method for diagnosis of G-I tract smooth muscle tumors and classification of leiomyosarcoma.

Cell Nucleus↗

Studies in longstanding ulcerative colitis with special reference to malignant transformation of the colorectal mucosa.

The risk of colorectal carcinoma was estimated retrospectively in a cohort of patients with ulcerative colitis from three defined geographical areas (West Midlands and Oxford regions, England and Stockholm County, Sweden). The cohort consisted of 824 primary referral patients with a diagnosis of ulcerative colitis established within five years from onset of symptoms between 1945-1965. All patients were 15 years of age or more at onset and they were followed for a minimum of 17 years and a maximum of 38 years. There was an eight-fold risk of developing colorectal cancer in the series as a whole, relative to that of the general population. In extensive colitis the risk was nineteen-fold. The cumulative risk of developing cancer in extensive colitis was 12% after 25 years of disease duration. No significant effects of the cancer risk for age at onset, sex or referral centre were detected. Abnormal, aneuploid DNA-content of cell nuclei in colorectal mucosal biopsies was found in five out of 53 patients with longstanding, total ulcerative colitis in a prospective study using flow cytometric DNA-analyses. Findings of DNA-aneuploidy were found at repeated examinations in four of these patients and there was a correlation with precancerous, mucosal changes (dysplasia) found at histological examination. In one patient DNA-aneuploidy preceded the finding of a carcinoma (Dukes' A) in the colon. Microspectrophotometry of imprint preparations from mucosal biopsies was compared to flow cytometry for detection of nuclear DNA-aneuploidy in seven patients with ulcerative colitis in a prospective study. DNA-aneuploidy was detected in five patients in eight separate locations of the colon and rectum. There was a good conformity between the two methods in the detection of DNA-aneuploidy, which was detected in non-dysplastic mucosa as well as in association with dysplasia. In a fifteen year follow-up surveillance program, comprising 72 patients with total ulcerative colitis, colonoscopy was performed at fixed intervals and biopsies sampled from ten predetermined locations in the colon and rectum. Definite dysplasia developed in 12 patients, two of which had carcinoma (Dukes' A). Nine patients were selected for prophylactic colectomy due to findings of dysplasia. A sequential development of dysplasia was found in seven patients. The cumulative risk of developing at least low grade dysplasia was 14% after 25 years of disease duration. Using flow cytometry, DNA-aneuploidy was detected in 12 out of 59 patients, significantly correlating with low and high grade dysplasia.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

Effect of beta-adrenoceptor stimulation or blockade on regional myocardial function and regional O2 consumption during myocardial ischemia.

The purpose of this study was to determine the effect of beta-adrenoceptor activation and blockade on the relationship between regional myocardial function and regional O2 consumption in ischemic and nonischemic myocardium. Myocardial regional segmental function was assessed in 28 open chest, anesthetized dogs using subepicardial dimension gauges. Ten min after LAD (left anterior descending coronary artery) occlusion, dogs were given i.v. saline, 2 mg/kg propranolol, 0.2 mg/kg pindolol, or 1 microgram/kg/min isoproterenol. Coronary blood flow was determined using radioactive microspheres before LAD occlusion, 10 min after occlusion, and 2 hr after LAD occlusion. Regional O2 consumption was determined using microspectrophotometry. LAD occlusion did not alter any index of myocardial function measured in the nonischemic region, but in the ischemic region, end systolic length (ESL) was increased 20% while shortening was converted to systolic bulging. No agent resulted in an improved ischemic regional function or an altered O2 consumption during LAD occlusion. In the nonischemic region the per cent shortening was increased 60% with isoproterenol compared to control. Propranolol and pindolol both increased the non-schemic regional ratio of per cent shortening vs O2 consumption significantly, suggesting an improved efficiency while isoproterenol lowered this ratio. When per cent shortening was plotted vs regional O2 consumption for all treatments, a significant linear relationship was observed in the nonischemic region. Thus, no drug treatment used in this study significantly improved central ischemic regional function, or O2 consumption, but both beta-adrenoceptor blockers seemed to result in an improved relationship between segmental shortening and O2 consumption in the nonischemic region.

Adrenergic beta-Agonists↗

X-chromatin (Barr bodies) correlated with the DNA content of lobular neoplasia and invasive lobular carcinoma in the female breast.

Literature relating X-chromatin with the histologic grade in carcinoma of the breast is briefly reviewed. X-chromatin incidence has been determined in 14 cases of lobular neoplasia and 5 invasive lobular carcinomas of the breast and has been correlated with the DNA content in these sections examined by Feulgen microspectrophotometry. In 11 of the cases of lobular neoplasia, the DNA values fell within the diploid range and in the 3 remaining cases within the diploid to tetraploid range, while the 5 invasive lobular carconomas showed a DNA distribution between the diploid and tetraploid ranges. The X-chromatin counts of these cases were variable but did not correlate with the cytophotometrically observed degree of malignancy in lobular neoplasia. No statistically significant differences in X-chromatin count of lobular neoplasia and invasive lobular carcinoma could be seen.

Breast Neoplasms↗

Collagen in human breast cancer: morphological and biochemical study.

The authors studied the distribution and the composition of collagen in neoplastic tissue from mastectomy specimens of 15 patients treated for an infiltrating ductal carcinoma NOS, in comparison to normal tissue of the opposite quadrant. The study was performed by light microscopy, microspectrophotometry, and electrophoretic methods. The results demonstrate a characteristic stromal organization of breast cancer in relation to collagen type I content.

Breast↗

Quantitative deoxyribonucleic acid analysis of patients with mild cervical atypia: a potentially malignant lesion?

Quantitative deoxyribonucleic acid (DNA) analysis of cervical biopsy material from 32 women with cytologic, colposcopic, and histologic evidence of mild cervical atypia consistent with cervical intraepithelial neoplasia I, reactive atypia, or human papillomavirus infection was carried out using flow cytometry and Feulgen microspectrophotometry. Evidence of aneuploidy, ie, neoplastic transformation, was demonstrated in all cases of cervical intraepithelial neoplasia I and 68% of cases with human papillomavirus-induced atypia. These results support the growing impression that human papillomavirus-induced cervical atypia should be regarded as a true precursor of cervical neoplasia, and emphasize the necessity to refer patients with mild atypia for definitive diagnosis and management.

Carcinoma in Situ↗

Diagnostic and prognostic impact of cytochemically assessed nuclear DNA contents in human adenocarcinomas of the mammary gland.

Cytochemical assessments of the nuclear DNA contents in carcinomas of the breast can be used for both prognostic and diagnostic purposes. Two main techniques are currently being used, viz. flow cytometry (FCM) and microspectrophotometry (MSP). An account of their advantages and disadvantages is given. In addition, an old, rather crude, cytophotometric technique can be used for histopathological sections of paraffin-embedded specimens. The principal sampling procedures are fine-needle aspiration biopsy and the so-called imprint technique, where the specimens are made from the cut surface of the freshly excised operation specimen. Paraffin-embedded histopathological material can also be used, applying a newly developed MSP procedure, where isolated nuclei from deparaffinized/disintegrated specimens are analyzed. Most important is that intact cell nuclei, representative for the whole tumour nodule, can be obtained. The simultaneous use of FCM and MSP is also of utmost importance for the reliability in the interpretation of the results. Then, a kind of "DNA malignancy grading" is obtained that in several investigations has proven itself to be an excellent prognosticating tool that can be used for making an adequate choice of therapy for the individual patient. The diagnostic value of the results of the cytochemically assessed nuclear DNA distribution patterns is not so high as the prognostic one. Tumours with a diploid type of nuclear DNA content can be found both among benign and malignant neoplasms. However, a neoplasm with an aneuploid DNA distribution pattern can almost certainly be considered highly malignant.

Adenocarcinoma↗

Prediction of metastasis of uveal melanoma: comparison of morphometric determination of nucleolar size and spectrophotometric determination of DNA.

We studied 95 cases of uveal melanoma using cytomorphometry to measure the standard deviation of nucleolar area (SD-NA) based on 200 cells per tumor and microspectrophotometry to determine the quantity of DNA in the nucleus of 100 cells per tumor. The 95 cases of uveal melanoma, in which the eye was enucleated between 1970 and 1973, were selected from the files of the REgistry of Ophthalmic Pathology. Forty-nine patients survived with a median follow-up of 15 years without evidence of metastasis at the time of the last follow-up, and 46 patients died of metastatic melanoma. Statistical analysis indicated that SD-NA was a better predictor of metastasis than DNA determination in this data set.

Cell Nucleolus↗

Absolute DNA values from Feulgen microspectrophotometric measurements and quantitative electron microscopy. A comparison of two species.

Quantitative electron microscopy (QEM) and microspectrophotometry were used to correlate the Feulgen stain absorption values to the calculated picograms of DNA. Measurements were made in human lymphocytes, rainbow trout lymphocytes and nuclei of trout erythrocytes. The median dry weight of the nucleus, as determined by QEM, was 35.9 pg for a human lymphocyte and 30.5 pg for a trout lymphocyte. Using Salzman's value of 20% DNA per chromosome (i.e., chromatin), a human lymphocyte nucleus thus contains 7.18 pg of DNA and a trout lymphocyte nucleus 6.1 pg of DNA. The mean Feulgen absorption value of the nucleus, given in arbitrary units (AU), was 14.5 for a human lymphocyte, 12.7 for a trout lymphocyte and 12.0 for a trout erythrocyte. From these values, it was derived that each picogram of DNA of a human lymphocyte nucleus is represented by 2.02 arbitrary Feulgen units while the values for trout nuclei were 2.08 AU and 1.97 AU. On the average, we find that each picogram of DNA is represented by two arbitrary Feulgen units in our microspectrophotometric measurements.

Animals↗