Existence of a polar digitalis-like factor in mammalian hypothalamus

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.     

A novel function of glutamine in cell culture: utilization of glutamine for the uptake of cystine in human fibroblasts

Transport and metabolism of glutamine has been investigated in human diploid fibroblasts, IMR-90. Glutamine was taken up via System ASC (Na+-dependent amino acid transport system especially reactive with short or polar side chain amino acids). In the routine culture medium the cells contained a large quantity of glutamate; its major source was shown to be glutamine in the medium. Previously we described a transport system that mediates the entrance of cystine in exchange for the exit of glutamate (Bannai, 1986). Since the cystine taken up is reduced to cysteine and the cysteine readily exits to the medium where it is oxidized to cystine, a cystine-cysteine cycle across the plasma membrane has been postulated. When the cells were cultured in glutamate/glutamine-free medium, intracellular glutamate decreased, depending on the amount of cystine in the medium; in the absence of cystine, glutamate decreased very slowly. When the cells were cultured in ordinary medium, glutamine in the medium decreased, and glutamate in the medium increased. Both changes were well correlated with cystine concentration in the medium. These results are consistent with the view that the intracellular glutamate, of which the source is glutamine in the medium, is released from the cells into the medium in order to take up cystine and thereby to rotate the cystine-cysteine cycle. In the routine culture one-third to one-half of the total consumption of glutamine seems to be used for the uptake of cystine.     

Immunocytochemical localization of cathepsins B, H, and their endogenous inhibitor, cystatin beta, in islet endocrine cells of rat pancreas

To determine the characteristics of lysosomes in rat islet endocrine cells, we examined the precise localization of cathepsins B, H, and L and their specific inhibitors, cystatins alpha and beta, using immunocytochemical techniques. By use of serial semi-thin sections, we detected immunoreactivity for cathepsin B in insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-positive (PP) cells. Strong immunoreactivity for cathepsin H was seen in A-cells and weak immunoreactivity in PP cells, but none in others. Immunodeposits for cystatin beta were demonstrated in B-cells. Brief dipping of thin sections in 1% sodium methoxide before the following immunocytochemical reaction enhanced specific deposits of immunogold particles on the target organelles. Use of a double-immunostaining technique showed co-localization of insulin with cystatin beta in many secretory granules. This suggests that cystatin beta may regulate converting enzymes participating in the maturation process of insulin. By use of an immunogold technique, heterogeneous localization of cathepsins B and H in lysosomes was also found among islet cells at the light microscopic level. This may be due to the difference in peptides degraded in lysosomes among the cells.     

Molecular cloning of a cDNA coding for 70 kilodalton subunit of soluble guanylate cyclase from rat lung

A complementary DNA clone corresponding to the 70 kDa subunit of soluble guanylate cyclase (EC 4.6.1.2) of rat lung has been isolated. The primary structure of the cDNA consisted of 3063 nucleotides including a 1857-nucleotide coding region for 619 amino acids, and the calculated molecular weight was 70476. Blot hybridization of total poly(A)+RNAs from rat tissues detected a mRNA of about 3.4 kilobases. The amount of mRNA was abundant in lung, cerebrum and cerebellum, moderate in heart and kidney, and low in liver and muscle. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated the presence of one gene in the rat haploid genome. The amino acid sequence of the 70 kDa subunit has partial homology with particulate guanylate cyclase from sea-urchin sperm, and protein phosphatase inhibitor I.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

A novel cell-surface antigen expressed on most leukocytes and a minor cortisone-resistant population of thymocytes in rats: characterization by monoclonal antibodies

A cell-surface antigen on rat lympho-hemopoietic cells was determined by using a monoclonal antibody, R2-1B3 (1B3). The 1B3 antibody, when tested for its reactivity with different hemopoietic cells by cytofluorography with a FACS analyzer, labeled more than 80% of lymph node, spleen, and bone marrow cells and 10-20% of thymus cells. Cytofluorographic analysis performed on purified rat T cells, B cells, macrophages, and granulocytes demonstrated that the antigen defined by 1B3 was readily detectable on all of these cell types, with the greatest expression on B cells. A minor population of thymocytes that were labeled by 1B3 appeared to be cortisone-resistant and were located mainly in the thymic medulla. These 1B3 positive thymic cells seemed to be functionally more mature than 1B3-negative thymus cells as suggested by the fact that the cytotoxic treatment of thymus cells with 1B3 antibody and complement (C) resulted in significant reduction of their responsiveness to phytomitogens and lymphokines derived from concanavalin A (con A) activated rat spleen cell cultures. Immunochemical data showed that 1B3 antibody recognized the broad ill-defined band with a molecular weight of 32K to 47K daltons as estimated by SDS-polyacrylamide gel electrophoresis. These data indicate that the 1B3 defined antigen is distinct from other, previously reported, antigens on rat lymphoid cells including leukocyte-common (L-C) and MRC OX-22 antigens, and that this 1B3 antibody is a useful reagent for analyzing the intrathymic differentiation of T cells in rats.     

Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.     

Strain-dependent differences in susceptibility of mice to experimental Angiostrongylus costaricensis infection

Experimental Angiostrongylus costaricensis infection was carried out in inbred strains of mice (C57BL/6 BALB/c, DBA/2 and C3H/He). All strains became infected with this parasite. Marked differences in mortality and in worm burden were found among inbred strains of mice tested. A significant reduction was shown in worm length from mice compared to that from cotton rats.     

Vegetative growth and productivity ofEichhornia azurea with special emphasis on leaf dynamics

The lifeform and the biological production of pure stands ofEichhornia azurea Kunth in three lakes in tropical Brazil were studied. The lifeform ofE. azurea is termed ‘semi-emergent’, because the plant has well developed trailing stems just under the water, and the aerial lamina emerges with the thick petiole. The density of shoot apices was 9.9, 17.2 and 17.1 m⁻² in Lake Dom Helvecio, Lake Jacaré and Lake Carioca, respectively. The mean daily increment of the apical shoot biomass was between 1.8 and 4.8 g m⁻² day⁻¹. The mean leaf life-span in Lake Dom Helvecio, Lake Jacaré and Lake Carioca was estimated to be 78, 49 and 64 days in the wet season and 73, 70 and 73 days in the dry season, respectively. The stem life-span was estimated to be about 28 months. Starch content in the current years' stem ranged from 24 to 118 mg g⁻¹ dry matter with fluctuations, the amplitude of which decreased with age. The differences for most of the growth parameters, such as density of shoot apices, daily increment of biomass and leaf life-span, between dry and wet season are smaller than those among the three lakes. Both the decrease in daily dry matter production and the increase in leaf life-span occurred in order from Lake Dom Helvecio to Lake Jacaré and Lake Carioca. The low productivity ofE. azurea is considered to be related to a low leaf area index, a long time interval for the emergence of new leaves, long leaf life-span and a low capacity for branching.