Melatonin Inhibits Mitotic Activity of Adrenocortical Cells In Vivo and in Organ Culture

The goal of this study was to examine the effects of melatonin, as well as those of melatonin and corticotropin (1-24 adrenocorticotrophic hormone (ACTH); Synacthen Depot) administered together, on the mitotic activity of adrenocortical cells in male and female mice. Melatonin was given subcutaneously once daily, in late-afternoon injections (between 16:00 and 18:00) in doses of 1 microgram, 10 micrograms, and 100 micrograms, and ACTH in a dose of 0.1 mg (10 U) daily for 10 days. Additionally, the highest dose of melatonin (100 micrograms daily) was administered together with ACTH. The metaphase-arrest technique using colchicine as a stathmokinetic agent was employed in the study. Melatonin, in all the examined doses, significantly decreased mean mitotic activity rate (MMAR) of the adrenal cortex in both male and female mice. Moreover, in a dose of 100 micrograms, melatonin suppressed the mitogenic effect of ACTH on the adrenal cortex. Furthermore, the present study investigated the effects of melatonin (5 x 10(-7)M), N-acetylserotonin (NAc-5HT) (5 x 10(-7)M), and ACTH (250 mU/ml or 1,000 mU/ml) alone as well as the effect of ACTH (250 mU/ml) applied jointly with melatonin on the mitotic activity of adrenocortical cells in rat adrenal explants incubated in vitro. Both pineal indoleamines (melatonin and NAc-5HT) significantly decreased the MMARs of adrenocortical cells. Corticotropin, as well as ACTH and melatonin applied together, also reduced the MMAR of adrenocortical cells. The present data suggest that melatonin may be directly involved in the inhibitory control of adrenocortical cell proliferation.     

Melatonin Inhibits the Basal and TSH-Stimulated Mitotic Activity of Thyroid Follicular Cells In Vivo and in Organ Culture

The aim of the present study has been to examine the effect of melatonin, administered to mice as daily subcutaneous injections for a total of 10 days, on the mitotic activity of thyroid follicular cells (TFC). The colchicine metaphase-arrest technique was employed in the experiment. We found that melatonin (10 micrograms and/or 100 micrograms injection) decreased significantly the mean mitotic activity rate (MMAR) of TFC in both male and female mice. Moreover, melatonin totally suppressed the stimulatory effect of TSH on the MMAR of TFC in both sexes of mice. Furthermore, the effect of melatonin (5 X 10(-7) M) on the proliferation of TFC in the organ-cultured rat and mouse thyroid explants was investigated. It was found that melatonin almost totally suppressed the MMAR of TFC in organ culture. Moreover, melatonin blocked the stimulatory effect of TSH on the MMAR of TFC in both rat and mouse thyroid explants. N-acetylserotonin (NAc-5HT, 10(-6) M) also decreased the MMAR of cultured thyroid explants, but its effect was less expressed when compared to melatonin inhibition. The present data indicate that melatonin can exert its inhibitory effect on the proliferation of TFC directly at the thyroid level, since this pineal indoleamine has been shown to suppress not only basal but also TSH-stimulated mitotic activity. The results are in agreement with the hypothesis of a pineal-thyroid negative feedback, assuming the direct inhibitory effect of melatonin on the thyroid growth.     

Neoplastic Lymphoid Reticulum Cells in the Peripheral Blood: A Histochemical Study

Abnormal appearing cells having the superficial appearance of largelymphocytes were found in some cases of generalized malignant proliferationsaf the reticulum cell system and were studied by cytochemical methods andby phase microscopy. They were found to be very similar in their reactions tothe "fixed" or tissue reticulum cells and in many respects to monocytes, butdiffered markedly from the cells of the lymphocytic system with which theyare often confused. The term neoplastic lymphoid reticulum cell is appliedto these cells since it indicates that they are malignant in nature and fundamentally of the reticulum cell type, although in fixed and stain material theymay show some of the morphologic characteristics of the lymphocytic cells.

Submitted on June 3, 1960 Accepted on October 14, 1960     

A New Method for Phenotyping Red Blood Cells Using Microplates

BACKGROUND AND OBJECTIVES: The supply of phenotyped red blood cells (RBC) for patients with several RBC antibodies presents a difficult task to hospital blood banks and regional blood centers. The aim of this study was to establish a low-cost typing system to allow extensive phenotyping of regular blood donors for clinically significant RBC antigens. MATERIALS AND METHODS: We developed a new buffer that greatly intensifies the antigen-antibody reaction and thus reduces the quantity of serum needed for phenotyping. The procedure was carried out on microplates. RESULTS: A total of 20,435 regular blood donors have been typed to date. For 752 units required for transfusion, 3,584 phenotyping tests were performed, validating the results by tube or gel typing methods; agreement was achieved in all cases. CONCLUSION: This technique seems adequate for phenotyping a large number of RBC units at very low cost, thus facilitating the availability of phenotyped blood.     

γ-Glutamyl Transpeptidase Activity in Liver of Alcoholics and Its Histochemical Localization

The activity and the histochemical localization of gamma-GTP in the liver of chronic alcoholics were investigated. Mean serum gamma-GTP activity in alcoholics was 542.5 +/- 337.9 milliunits/ml, and that of patients with nonalcoholic liver disease was 34.3 +/- 22.6 milliunits/ml. Hepatic gamma-GTP activity in alcoholics was significantly increased compared to that in control patients (15.62 +/- 9.29 versus 4.04 +/- 2.67 units/g of liver; p less than 0.001). A significant correlation was observed between hepatic and serum gamma-GTP activity. Light microscopically, a marked gamma-GTP activity was found in the bile canaliculi and a diffuse activity in the cytoplasm in alcoholic livers. By contrast, in the livers of nonalcoholic patients, only slight activity was observed in the bile canaliculi. The electron micrographs showed the enzyme was localized in the microvilli of both the bile canalicular and plasma membranes and the endoplasmic reticulum near the mitochondria in alcoholics. But a very low activity was demonstrated in the plasma membranes in the livers of nonalcoholic patients.     

Biological Activity of a Growth Factor for Ovarian Cells

The biological activity of a protein growth factor is described. This factor was isolated from bovine pituitary tissue, and its activity was tested on cells of rat ovarian origin. Activity was assayed as growth-promoting potential measured by cell counts and is concentrationdependent. Similar growth stimulation was not produced by several cyclic nucleotides tested, neither was there crossreactivity between this growth factor and a chemically similar one produced by rat liver cells.     

A Diffraction Method for Measuring the Average Volumes and Shapes of Red Blood Cells

An instrument and technique are described for determining MCV, MCD and D/T from the visual measurement of the diffraction rings produced by red cells; MCV from the unfixed spherical cells and MCD from the fixed discoidal cells. The method eliminates a serious error due to asymmetry in retinal sensitivity near the fovea. The accuracy of the measurements is assessed from the repeatability of the observations of individual observers, and from the agreement between those of different observers. The validity of the optical theory in the deduction of the cell dimensions from the diffraction measurements is confirmed in the case of spherical cells: (1) By the agreement found between the values of MCV obtained with the spherocytometer on the one hand, and with the electronic counter and centrifuge on the other. (2) By showing that the constants for the various diffraction rings produced by spherical cells have their theoretical values. However, the constants found for the rings produced by the fixed discoidal cells differ significantly from their theoretical values. Because of this, and because of the shrinkage of the fixed cells, the values for MCD and D/T are only relative. The same criticism applies to the standard method for determining these data from dried blood films; but in dried films the shrinkage is variable, whereas in the new method described the shrinkage is constant. Error arising from the intensity of light in a diffraction ring being proportional to the fourth power of the diameter of the cell was examined and found to be negligible in the case of D/T, and to reach a significant amount in MCV only when anisocytosis is extreme. The possibility is considered that in abnormal cells variable shrinkage due to fixation may produce spurious variations in D/T. However, low values for D/T are found in spherocytosis, and high values in thalassaemia, in which the cells are flattened. Further, measurements of unfixed discoidal cells suspended in their own serum gave values of D/T closely parallel to those derived from the fixed discoidal cells. Results may be misleading when a microcytic or macrocytic anaemia is responding to treatment, or following transfusion. The error can be recognized and, by centrifugation, the cells present before treatment can be separated from the newly formed cells and measured in the ordinary way.     

食管癌变过程中细胞增殖活性的研究

肿瘤细胞的增殖率是决定肿瘤生物学行为的重要参数。本实验用ＬＳＡＢ免疫组织化学方法研究了食管上皮从正常──单纯增生──不典型增生──原位癌──浸润癌多阶段过程中ＰＣＮＡ表达情况和ＰＣＮＡ表达与肿瘤分化程度的关系，并与ＡｇＮｏＲｓ计数进行直线相关分析。结果表明：ＰＣＮＡ标记指数和ＡｇＮｏＲｓ与癌分化程度间均存在反变关系，前者有统计学意义，后者则无。二项指标间有较好的直线正相关。癌前各阶段病变ＰＣＮＡ标记率依次增高，各级之间差异显著，可为筛选癌前病变和发现早期癌制定一个较客观的量比标准。     

Method for Processing Leukocyte- and Platelet-Poor Red Cells in Closed Bags

If leukocyte- and platelet-poor red cells have been processed and stored in closed bags, their delivery, especially on holidays, will be easy. The modified warm-centrifuge method was applied to a quadruple bag prepared for such purpose. Red cells were processed in the first bag by the centrifugation of whole blood. The supernatant plasma was transferred to the second bag. A phosphate buffer in the third bag was introduced into red cells and diluted red cells were then incubated at 37 degrees C for 1 h in an inverted position. After centrifugation, the red cell phase below the buffy coat layer was isolated using a Biotest separation apparatus and stored in the fourth bag (containing a preservative with adenine, phosphate, glucose and 0.9% NaCl solution) for 28 days at 4 degrees C. Ninety-eight percent of the leukocyte and 97% of platelets in whole blood unit were removed with a red cell recovery of 86%. Biochemical changes in stored red cells were similar to those of conventional buffy coat-removed red cells suspended in the same preservative, except for rapid decreases in 2,3-diphosphoglycerate levels. Since processing buffy coat-removed red cells containing few leukocytes and platelets with the present method was simple and these units might be stored for over 14 days with minimum cell damage, we suggest that the present method is useful way of processing leukocyte- and platelet-poor red cells in blood centers.     

A Histochemical Method for the Demonstration of Leucocyte Naphthylamidase Using Two Substrates: Alanyl- and Methionyl-4-Methoxy-2-Naphthylamide

Histochemical methods for the demonstration of naphthylamidase* in tissues were based on the coupling properties of 2-naphthylamine (Burstone and Folk, 1956; Nachlas, Crawford and Seligman, 1957) after enzymatic release from amino acid naphthylamides. But significant diffusion of the amine and the lack of a suitable diazonium salt to achieve a sufficiently prompt capture reaction to form an insoluble dye as well as prolonged incubation prevented adequate histochemical localization. When the coupling rate of 2-naphthylamine was found to be significantly enhanced by the presence of a methoxy group in the 4-position (Nachlas, Goldstein, Rosenblatt, Kirsch and Seligman, 1959), l -leucyl-4 -methoxy-2 -naphthylamide (Rosenblatt, Nachlas and Seligman, 1958) was synthesized and found to serve as a more satisfactory substrate for naphthylamidase demonstration (Nachlas, Morris, Rosenblatt and Seligman, 1960). Replacement of the leucyl group in leucyl-2 -naphthylamide by alanyl or methionyl groups resulted in a more rapid hydrolysis by naphthylamidase (Nachlas, Goldstein and Seligman, 1962; Smith, Kaufman and Rutenburg, 1965; Monis, Wasserkrug and Seligman, 1965). It therefore appeared likely that the substrates alanyl- and methionyl-4 -methoxy-2 -naphthylamides would yield further improvement of localization of enzymatic sites and would be potentially useful for the demonstration of naphthylamidase in tissues such as white cells which are known to have a low content of this enzyme. In this report we present the data on the use of both substrates for the demonstration of naphthylamidase in human leucocytes.     

A Histochemical Method for the Demonstration of Leucocyte Naphthylamidase Using Two Substrates: Alanyl- and Methionyl-4-Methoxy-2-Naphthylamide

H istochemical methods for the demonstration of naphthylamidase* in tissues were based on the coupling properties of 2-naphthylamine (Burstone and Folk, 1956; Nachlas, Crawford and Seligman, 1957) after enzymatic release from amino acid naphthylamides. But significant diffusion of the amine and the lack of a suitable diazonium salt to achieve a sufficiently prompt capture reaction to form an insoluble dye as well as prolonged incubation prevented adequate histochemical localization. When the coupling rate of 2-naphthylamine was found to be significantly enhanced by the presence of a methoxy group in the 4-position (Nachlas, Goldstein, Rosenblatt, Kirsch and Seligman, 1959), l -leucyl- 4 -methoxy- 2 -naphthylamide (Rosenblatt, Nachlas and Seligman, 1958) was synthesized and found to serve as a more satisfactory substrate for naphthylamidase demonstration (Nachlas, Morris, Rosenblatt and Seligman, 1960).
Replacement of the leucyl group in leucyl- 2 -naphthylamide by alanyl or methionyl groups resulted in a more rapid hydrolysis by naphthylamidase (Nachlas, Goldstein and Seligman, 1962; Smith, Kaufman and Rutenburg, 1965; Monis, Wasserkrug and Seligman, 1965). It therefore appeared likely that the substrates alanyl- and methionyl- 4 -methoxy- 2 -naphthylamides would yield further improvement of localization of enzymatic sites and would be potentially useful for the demonstration of naphthylamidase in tissues such as white cells which are known to have a low content of this enzyme.
In this report we present the data on the use of both substrates for the demonstration of naphthylamidase in human leucocytes.     

Eosinophilic Leukemia A Morphologic and Histochemical Study

Correlated histochemical, phase and electron microscopic studies were employed in examining the eosinophils from a patient with acute eosinophilicleukemia. Numerous morphologic alterations were observed in the leukemiceosinophils and eosinophilic myelocytes. These alterations included asynchronous nuclear-cytoplasmic maturation; an increase in cell size; the formationof eosinophilic granules which vary markedly in number, size, contour, anddensity; and the presence of fibrillar formations in some of the leukemic cells.Histochemically, the major alterations observed in the leukemic cells werethe extensive deposition of glycogen in the cytoplasm and the demonstration of increased phosphorylase activity in these cells. Other minor variationsin the histochemical reactivity of the leukemic eosinophils also have beendescribed. Histochemical procedures included technics for proteins, aminoacids, carbohydrates, hydrolytic and oxidative enzyme activities.

Submitted on November 15, 1963 Accepted on January 30, 1964     

Thymidine Kinase Activity in Human Bone Marrow Cells

The thymidine kinase activity per 10⁶ DNA-synthesising marrow cells and the rate of incorporation of tritiated thymidine into the DNA of 10³ DNA-synthesising marrow cells were estimated in 9 haematologically normal patients and 49 patients suffering from a variety of haematological disorders. Slight increases in thymidine kinase activity were found in 6 of the 31 patients with haematological diseases associated with normoblastic erythropoiesis and greater increases were found in 3 of the 18 patients with megaloblastic haemopoiesis due to vitamin B₁₂ or folate deficiency. In the latter group, there was a statistically significant inverse correlation between haemoglobin levels and thymidine kinase activity. No correlation was found between thymidine kinase activity and the rate of incorporation of tritiated thymidine in either the normoblastic or megaloblastic group, suggesting that the level of thymidine kinase activity does not limit the rate of incorporation of exogenously supplied thymidine into the DNA of human bone marrow cells.     

Increased Natural Killer Cells Activity in Schizophrenic Patients

Background: Schizophrenia has been associated with altered immunity. Different studies regarding natural killer cell activity (NKA) in schizophrenic patients have shown inconsistent results. Objectives: To evaluate NK cell activity in schizophrenic patients in comparison with healthy control individuals.   Methods: 30 medication-free schizophrenic patients and 41 healthy sex, age and smoking status matched individuals were included in this study. NK cell activity of case and control subjects was measured by Methyl-Thiazol-Tetrazolium (MTT) test. Statistical analysis of the data was done using SPSS 11.5 software.   Results: NK activity of patients and normal subjects had a mean of 36.94 ± 26.15 (Mean ± SD) and 22.31 ± 17.92, respectively. A significant increase in NK activity in schizophrenic patients compared to controls (P = 0.011). Among patients, NK activity of smokers was significantly lower than that of non-smokers (P = 0.02). Other demographic factors didn't show any influence on NK activity.   Conclusion: The higher activity of NK cells in the schizophrenic patients as compared with the control population could explain the low incidence of cancer in these patients. Decreasing the effect of smoking on NK activity in the patients could be one of the responsible factors for the inconsistency in the results of different studies.     

Tissue Factor Activity of Normal and Leukemic Cells

Generation of procoagulant activity wasstudied in leukocytes and lymphocytesfrom normal human peripheral blood, leukocytes from patients with chronic myeloidleukemia (CML), chronic lymphatic leukemia (CLL), rabbit peripheral leukocytes,and macrophages harvested from the peritoneal cavity of rabbits. Marked procoagulant activity developed in peritonealmacrophages and rabbit peripheral leukocytes when incubated with endotoxin.Human peripheral blood leukocytes andlymphocytes also generated procoagulantactivity. When endotoxin was omitted theprocoagulant activity generated was considerably (3-12 times) lower. Leukocytesfrom patients with CML were less activethan normal leukocytes. Leukocytes frompatients with CLL generated very littleprocoagulant activity even after prolonged incubation. An antibody to rabbittissue factor neutralized the tissue factoractivity generated by rabbit leukocytes.

Submitted on January 12, 1973 Revised on March 30, 1973 Accepted on May 4, 1973     

Histochemical Study of Phosphorylase in Proliferating Cells of Intestinal Metaplasia and Carcinoma of the Human Stomach

A morphologic histochemical study of phosphorylase was carried out to investigate the relationship between gastric carcinoma and intestinal metaplasia. Intense phosphorylase activity was observed in the carcinoma cells, especially in well-differentiated adenocarcinoma, and in the proliferating cells of some intestinal metaplasias. Metaplastic epithelium other than the proliferating cells occasionally showed a positive reaction. Phosphorylase was negative in normal gastric epithelium, even in its proliferating cells. There was an apparent coincidence between the location of well-differentiated adenocarcinoma and the distribution of intestinal metaplasia, with the proliferating cells showing positive reaction for phosphorylase. These data suggest that the relationship between the proliferating cells of intestinal metaplasia showing phosphorylase activity and well-differentiated adenocarcinoma is apparently closer than the much-debated relationship between the epithelium of intestinal metaplasia and gastric carcinoma.     

用组织化学方法鉴别日本血吸虫细胞来源的研究

目的　探讨用组织化学方法鉴别血吸虫体外培养细胞来源或种类的可能性。　方法　取日本血吸虫肝期童虫,经酶消化处理后取混合细胞涂片,用成虫制作组织切片。将细胞涂片和组织切片分别平行作高碘酸希夫氏(PAS)、硫堇 (Thionin)、嗜银反应(根据Grimelius)、苦味酸 酸性品红(根据VanGieson,VG)、甲苯胺蓝(Tolui dineblue,TB)和苏木素伊红(HE)6种组织化学方法染色。光镜下比较观察,判定结果。　结果　PAS染色的大细胞呈亮红色与组织切片中卵黄腺着色接近,表明着色的大细胞来源于卵黄腺;含特有尼氏体的神经细胞胞浆被硫堇染色呈深蓝色 ;Grimelius染色的细胞和消化道上皮细胞均呈黑色;VG染色,部分细胞和虫体肌纤维组织均呈黄色;TB染色,细胞涂片仅见1个紫红色细胞,组织切片未见相同颜色的组织和细胞。　结论　PAS、硫堇、Grimeliu s、VG、TB和HE 6种组织化学染色,对体外培养的血吸虫细胞的显色特征与虫体相应器官或组织细胞的化学染色定位相一致。本法可用于鉴别血吸虫体外培养细胞的来源或种类,且操作简便、快速。     

A Method for the Positive Identification of Erythropoietic Cells in Chromosome Preparations of Bone Marrow

Chromosome preparations from the bone marrow of four patients with chronic granulocytic leukaemia were made after the cells had been cultured for 12 hours with iron isotopes in a medium with added GSH, ATP, cysteine and ascorbic acid, which permitted optimal iron uptake and incorporation. The cells were fixed for 15 minutes only in formaldehyde-methanol-acetic acid. Auto-radiographs of these preparations showed that the Ph1 chromosome was present in erythroid cells. This indicates that erythroid and myeloid cell lines have a common stem cell in the bone marrow.     

A Method for Detecting Factor-VIII Clotting Activity Associated with Factor VIII-related Antigen in Agarose Gels

A method for detecting factor-VIII clotting activity in agarose is described. It is based on factor VIII promoting coagulation in a mixture of haemophilic plasma in agarose which is detected by a change in opacity. When this test was used to detect factor-VIII clotting activity in a one-dimensional Laurell electroimmunoassay for factor VIII-related antigen all the factor-VIII activity was found in the same position as the factor VIII-related antigen immunoprecipitate. Factor-VIII clotting activity did not appear to be simply trapped in this immunoprecipitate and therefore it has been concluded that the molecule containing factor-VIII clotting activity carries factor VIII-related antigen determinants.