Influence of ascorbic acid, sodium citrate, and sodium bicarbonate on the uptake of 59Fe-transferrin, 54Mn-transferrin, and 65Zn-transferrin from lactating mouse mammary gland cells.

The effects of ascorbic acid, sodium citrate, and sodium bicarbonate on 59Fe-transferrin, 54Mn-transferrin, and 65Zn-transferrin uptake by the receptors disposed of plasma membrane isolated from lactating mouse mammary gland cells have been investigated. The effect of 10(-2) mol/L ascorbic acid alone and in combination with NaHCO3 on the 59Fe-transferrin uptake is significant and positive. 54Mn-transferrin and 65Zn-transferrin binding to the cell receptors are influenced optimally by 0.5 mol/L sodium bicarbonate. Sodium citrate alone or in combination with other substances always has a negative effect on binding of these three metals. It is suggested that a precise mechanism may exist with large possibilities to rearrange metal uptake and its transport from blood to milk.

Effect of bovine milk antigens and egg lysozyme on the binding of 59Fe-lactoferrin to platelet plasma membranes.

1. Platelets bind specifically to lactoferrin. A significant similarity between human lactoferrin and some bovine milk proteins has been established. 2. Because of the structural homology of lactoferrin and cows milk proteins they are able to influence lactoferrins regulatory function on the level of its binding to membrane receptors on platelets. 3. An inhibitory effect of bovine alpha-lactalbumin and of beta-lactoglobulin on lactoferrin-receptor interaction was shown. 4. Bovine alpha-lactalbumin competes with lactoferrin for the binding sites. 5. Scatchard plot analysis of data shows one binding site for lactoferrin in the presence of alpha-lactalbumin with an affinity constant, Ka = 0.46 x 10(9) mol/l and 335 receptors/cell. 6. The inhibitory effect of beta-lactoglobulin reaches 62% and is different for the common fraction beta-lactoglobulin and the genetic variants beta-lactoglobulin A and B. 7. beta-lactoglobulin does not compete with lactoferrin for the membrane receptors. 8. Bovine casein and egg lysozyme stimulate 59Fe-lactoferrin binding to the receptors. The mechanism of these effects is still unknown. 9. Tested alimentary antigens are able to interact with lactoferrin and also with some platelet membrane structures. 10. Established changes in lactoferrin binding to the platelet membrane might be in relation to lactoferrins regulatory function and (or) eliminating mechanisms of these alimentary antigens.

Competition of Mn2+ and Zn2+ with 59Fe2+ and 59Fe3+ for the plasma membrane receptors from lactating mouse mammary gland.

Nonlabeled MnCl2 and ZnSO4 compete with 59Fe(2+)-ascorbate and 59Fe2(3+)O3 for transport binding sites situated on the plasma membranes of lactating mouse mammary gland cells. The binding was found to be a process reaching saturation. The heterologous competition used here ruled out the participation of transferrin and to propose that Fe, Mn, and Zn are transported from blood to milk by a mechanism involving one receptor during lactation. Further experiments are necessary to establish the details of the transport mechanism.

Effect of insulin and adrenaline on the 59Fe transferrin uptake of lactating mouse mammary gland cells.

The results of insulin action (0.4 IU per mouse) are demonstrated in intact animals only. This action leads to a higher uptake of 59Fe. rabbit transferrin of isolated cells from lactating mouse mammary gland. It is suggested that most inactive transferrin receptors in the cell membrane are incorporated by the hormone action or some new receptors are synthesized. On the contrary, adrenaline in a dose 0.5 micrograms per animal demonstrated an opposite effect--a lower uptake of 59Fe. human transferrin from lactating mouse mammary gland. This is probably due to a redistribution of some part (about 28%) of the iron. Instead of flowing to the mammary gland it flows towards other organs for overcoming the stress situation. An alternative explanation could be the inhibition of endogenous insulin secretion by adrenaline. From our data it follows that insulin and adrenaline have an antagonistic effect on regulation of Fe transport in lactating mouse mammary gland.

Binding of mouse and rabbit iron-59 transferrins to lactating mouse mammary epithelial cells.

The binding of mouse and rabbit transferrins to lactating mouse mammary epithelial cells was tested in a 59Fe-protein-binding assay. The homologous and heterologous binding was slow during the first 30 min, after which the uptake steadily increased. In ligand concentration-dependent saturation studies, the heterologous rabbit protein showed a high degree of binding and required approximately 9.7 ng of ligand to saturate approximately 2 x 10(6) cells. The homologous mouse protein demonstrated a low degree of binding and failed to demonstrate saturation at the above ligand concentration. Scatchard plot for homologous binding data was nonlinear and implied a low (1.08 x 10(-10) M) and a high (1.82 x 10(-9) M) affinity interaction mechanism. However, the plot for heterologous binding was linear and characterized by one high affinity (1.0 x 10(-9) M) binding interaction. A total of 11,000 and 19,600 binding sites per cell were estimated for mouse and rabbit proteins, respectively. These data suggest a binding crossreactivity between mouse and rabbit transferrins. A high affinity binding mechanism seems to be conserved in proteins from both species; however, an additional low affinity binding was present only in the homologous system.

Effect of ascorbic acid, sodium citrate, sodium bicarbonate, and their combination effect on the binding of 59Fe2+ and 59Fe3+ to plasma membrane of lactating mouse mammary gland.

Isolated plasma membranes of lactating mouse mammary gland were treated with different concentrations of ascorbate, sodium citrate, sodium bicarbonate, and combinations of them (from 16 x 10(-10) to 4 x 10(-6) moles/L) and studied for the binding of 59Fe2+ and 59Fe3+ at pH 7.4. The results show that the Fe3+ form of iron is under a greater influence of anions used in these experiments. The Fe2+ form of iron is weakly bounded and affected. It is suggested that the form with a greater positive electric charge is more effectively bound to the receptors in plasma membranes.

Lactating mouse mammary cell uptake of 59Fe-transferrin. The effect of KCN, 2,4-dinitrophenol, ethanol and ATP.

1. Amounts of KCN of 0.2, 1.0 and 2.0 X 10(-11) mol in a probe do not prevent the uptake of transferrin iron from lactating mouse mammary gland cells. 2. The effect of 5, 10 and 100 X 10(-11) mol in a probe 2,4-dinitrophenol dissolved in 20% ethanol is also negative on the iron-transferrin uptake from the cell. 3. There is a statistically significant decrease of uptake of transferrin-iron under the influence of 20% ethanol in the incubation mixture (25, 50 and 100 microliters). The effect of ethanol is diminished by 2,4-dinitrophenol in the solution. 4. The uptake of 59Fe-transferrin from cells of lactating gland was blocked by ATP (in amounts 0.5, 1.0 and 10.0 X 10(-11) mol per probe) while other concentrations have no effect. It seems that ATP does not release 59Fe from transferrin, nor help the iron uptake from the cells. 5. All evidence indicates that the uptake of transferrin-iron from lactating mammary gland cells is not an energy dependent process.

A program package for self-assessment and examination in biochemistry.

A BASIC program has been developed for self-evaluation and testing of an individual's knowledge of main categories in biochemistry. The computer tests permit sequential or random distribution of items, random distribution of answers in each item at each new run, effective feedback control with the student, use of a program clock to determine different time allowance, precise and quick analysis and grading of student's answer thus saving the time and labour of examiners. Editing, deleting and appending of new items is easily achieved. Information is obtained for verifying the quality of the test itself. Examination versions of tests in ten categories have been combined into an annual computer test in biochemistry.

The difference between cytosol and membrane growth-related protein kinase activities in lactating mouse mammary gland.

1. A protein kinase activity which is cAMP-independent, inhibited by the bioflavonoid quercetin and probably connected to the growth of mammary gland cells was isolated and partially purified from cytosol. 2. Another protein kinase activity was demonstrated in crude membranes of lactating mouse mammary gland. 3. By the use of several different synthetic peptides as a substrate, it was demonstrated that the cytosol enzyme was a serine kinase, while the membrane protein kinase activity was mainly due to tyrosine kinase.

Opiate receptor binding in the brain of rat during stress.

Two types of opioid receptors were studied in the brain of rats: Delta (for endogenous opiate) and mu (for exogenous opiates). 3H derivates: D-Ala2-enkephalin and Naloxone were used as labeled ligands. The results obtained were calculated by computer program for automatic estimation of the data using approximation equations. An increase of binding delta receptors is observed in both types of stress (2-8 times), while to the mu receptors the binding is less effective mainly after irradiation. These data suggest that a close interaction exists between sympathoadrenal system and opioid mechanisms during stress.

The effect of zinc on [59Fe]lactoferrin binding to blood neutrophilic leucocytes and colostral cells.

Zn2+ inhibits the binding of [59Fe]lactoferrin to neutrophilic leucocytes. The inhibiting effect is proportional to zinc concentration in the range 10-330 mumol/l. Zn2+ inhibits the [59Fe]lactoferrin binding to the colostral cells in the same degree as PMN. The inhibiting effect of Zn2+ on [59Fe]lactoferrin binding to neutrophilic leucocytes is equal to those of non-labelled lactoferrin and transferrin. Fe2+ and Cu2+ does not have such effect on binding of [59Fe]lactoferrin to the PMN leucocytes.

Angiotensin II receptors in the brain and adrenal glands of rats with experimental and spontaneous hypertension.

Angiotensin II receptors in the brain and adrenal glands of rats with experimental and spontaneous hypertension were studied. The data have shown that the capacity of binding sites does not change substantially in the adrenal gland while in the brain of spontaneous hypertensive rats it is lower and increases in those with experimental hypertension. There is also a change in the affinity of binding in both the adrenal gland and the brain of the spontaneously hypertensive rats. The data suggest some changes in tissue sensitivity to hormone in the spontaneously hypertensive rats.

Insulin receptor in cells of two transplanted tumors of mouse mammary gland.

Highly specific insulin receptors have been identified in the cells of two transplanted tumors of mammary gland in mice. The two tumors used for transplantation had morphological characteristics of an undifferentiated adenocarcinoma with a different growth rate. The specific binding was proportional to the number of tumor cells in the incubation mixture. Saturation of insulin binding sites was observed when the concentration of 125I-insulin increased over 5 X 10(-9) moles/l. The specificity of insulin binding to tumor cells was examined by means of competition with insulin derivatives: des-octapeptide insulin, tert-butyloxycarbonyl3-insulin (BOC3) and its des-octapeptide derivative were without any detectable activity in the concentration range used, i.e. from 5.5 X 10(-11) to 5.5 X 10(-5) moles/l. The data obtained from Scatchard's analysis of the binding were not linear and the affinity constants were similar for the cells of the two tumors. The total number of insulin receptor sites per cell, estimated by linear regression analysis, were higher for the cells isolated from the slow-growing tumor as compared to that of cells isolated from the fast-growing tumor and a lactating mammary gland. These results point to the possibility that the growth of mammary tumors could be under insulin regulation via insulin receptors.

Lactoferrin binding to neutrophilic polymorphonuclear leucocytes.

Neutrophilic polymorphonuclear leukocytes (PMN) bind specifically lactoferrin. This binding was characterized by two types of binding sites: with higher affinity and low capacity and another site with lower affinity and higher capacity (respectively Kaff 1 = 2.2 X 10(9) M-1 and about 39,000 binding sites per cell and Kaff2 = 0.6 X 10(9) M-1 and 76,000 binding sites per cell). The lactoferrin binding to PMN leucocytes depends on the concentration of labelled lactoferrin, the number of cells and the time for incubation. The presence of lactoferrin receptors on the PMN leucocytes is connected most probably with its effect on the cell function of these cells.

Binding of transferrin-iron to the plasma membrane of a lactating rabbit mammary gland cell.

1. Cells isolated from a lactating rabbit mammary gland have been investigated for transferrin-iron receptors. The existence of these structures has been demonstrated through a specific binding with a competitor non-labelled rabbit transferrin. 2. The interaction of iron-receptor complex depends on the concentration of [59Fe]transferrin, the number of cells and the time. 3. Scatchard's plot of data indicates two classes of receptor sites: one with a binding capacity 6.48 x 10(-9) g Fe per cell and affinity constant 2.48 x 10(10) M-1 and another with 1.06 x 10(-8) g Fe per cell and 4.66 x 10(11) M-1 respectively. 4. The probable mechanism of the iron transport from blood to milk through the lactating cell was discussed.

[Insulin receptors in isolated cells of murine lactating mammary gland]. / Insulinovye retseptory v izolirovannykh kletkakh laktiruiushchei molochnoi zhelezy myshei.

Insulin receptors in isolated cells from lactating mouse mammary gland were investigated by 125I-labeled insulin. A modified chloramin T-method after Hunter and Greenwood (1962) was used for insulin labelling with 125I; 125I-insulin with a high specific activity (80--120 muCi per microgram), high immunoreactivity and preserved biological properties was obtained. Three methods of labeled insulin purification were also studied: absorption on the cellulose column, gel chromatography on different types of Sephadex (G-25, G-75, G-100) and electrophoresis on polyacrylamide gel. The best conditons for iodination and purification of 125I-insulin suitable for studying the receptor-insulin binding in the isolated cells from mouse mammary gland were selected. Insulin binding to its receptor was found to be a specific reversible process, having high affinity and a tendency to saturation. The receptor-insulin-binding equilibrium was reached in 30 min at 24 degrees C. Specific binding was observed at a very low concentration of 125I-insulin (0.4.10(-9)M), close to the physiological level. Saturation was observed at a concentration of over 1.5.10(-9)M. Native, non-labeled insulin, at a concentration of 1 ng per ml lowered the labeled insulin binding by 10--20 per cent, whereas 10 ng per ml led to a 60 per cent lowering. The affinity constant of the process was about 10(9).M-1. Each cell had about 3 000 to 4 000 insulin binding sites.

Optimum conditions for the binding of insulin to its receptor.

The specific binding of insulin to the membranes from lactating mouse mammary gland was studied as a model of hormonereceptor type of binding. The basic ingredients of binding, the concentration of receptor protein and the concentration of labeled insulin were mainly studied. The characteristic changes in specific binding were followed, the adequate regression equation was draen and the optimum conditions of binding were established for further experiments. The expediency of applying shortened orthogonal plans, regression and analysis and graphic conture analysis were proved.

Radioimmunoassay of alpha-fetoprotein in the serum of patients with leukemia and malignant melanoma.

A sensitive double antibody radioimmunoassay has been used to measure Alpha-fetoprotein in the serum of healthy subjects, pregnant women and patients with a variety of malignant diseases including leukemia and melanoma. Elevated serum Alpha-fetoprotein levels were found in 2 of 35 patients with leukemia, 2 of 10 with melanoma. All the pregnant women studied had raised levels.