Specific elimination of cytotoxic cells. II. Reduction of cytotoxic activity by adsorption on monolayers results from removal of cytotoxic cells and not from inhibition of their activity.

Cytotoxic lymphocytes produced by in vitro sensitization to H-2 alloantigens may be adsorbed efficiently and specifically on monolayers of spleen lymphocytes attached to poly-l-lysine-coated polystyrene. Greater than 16-fold median reduction in activity resulted from adsorption to the target-type monolayer while adsorption to the attacker-type monolayer resulted in a less than two-fold median reduction. Since this reduction could have resulted from competitive inhibition by detached monolayer cells rather than from sticking of the cytotoxic cells to the monolayer, experiments were performed to distinguish between these possibilities. The inhibitory properties of a nonadherent population were tested directly by addition to a different attacker-target combination in which its killing properties were irrelevant but in which the H-2 genotype of detached monolayer cells was appropriate to cause competitive inhibition. No significant inhibition was observed; and this indicated that reduction of activity in an adsorbed population results from removal of the cytotoxic cells and not from inhibition by detached monolayer cells competing with the labeled targets for attackers.     

Identification of gastric cyclic AMP binding proteins

The photoaffinity ligand 8-azidoadenosine 3',5-monophosphate was employed to label cAMP binding proteins in both fractionated and freeze-thawed rabbit gastric glands. Fractionated glands incorporated the azido-cAMP label primarily into two cytosolic proteins with apparent molecular weights of 58 000 and 48 000. No enrichment of label was found in fractions containing basolateral or apical membranes. DEAE-cellulose chromatography of the cytosolic proteins resulted in the separation of two cAMP-dependent protein kinase peaks. Azido cAMP labelling of each peak suggested the initial peak contained type I cAMP-dependent protein kinase while the second peak contained the type II kinase. Labelling of 'resting' gastric glands resulted in radioactive proteins of apparent molecular weights of 58 000 and 48 000. When gastric glands were stimulated to produce acid by the addition of 10(-4) M histamine or 1 mM dibutyryl cAMP there was 32-44% dimunition of ligand incorporation compared to control glands. The results strongly suggest that histamine-mediated stimulus-secretion coupling in gastric glands involves activation of parietal cell cAMP-dependent protein kinases.     

应用气-质联用仪测定三种螺旋藻中的甾醇成分

应用GLC/MS联用仪对室内培养的钝顶螺旋藻(Spirulina platensis (Nordstedt) Geitler)、极大螺旋藻(S.maxima (Stechell & Gardiner) Geitler)和盐泽螺旋藻(S.subsalsa Oerst)的甾醇成分进行了测定。从钝顶螺旋藻和盐泽螺旋藻中共分出11个相同的甾醇组分:胆甾醇、胆甾烷醇、芸苔甾醇、麦角甾醇、海绵甾醇、菜子甾醇、豆甾醇、24-乙基-Δ~(5,7,22)-胆甾醇、β-谷甾醇、异岩藻甾醇和4α,23,24-三甲基Δ~(5,22)-胆甾醇;从极大螺旋藻中只分离出8个甾醇组分。其中胆甾醇含量最高。4α,23,24-三甲基-Δ~(5,22)-胆甾醇为蓝藻中首次报导。     

Spatial and temporal patterns of intracellular calcium in colonic smooth muscle

Summary Intracellular calcium [Ca²⁺] _i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca²⁺] _i transient followed by a steady-state increase as the characteristic [Ca²⁺] _i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca²⁺] _i signal in single cells. The distribution of [Ca²⁺] _i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca²⁺] _i present in the subplasmalemmal space and in one cell pole. [Ca²⁺] _i gradients within these regions were not constant but showed temporal changes in the form of [Ca²⁺] _i oscillations and spatial changes in the form of [Ca²⁺] _i waves. [Ca²⁺] _i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca²⁺] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca²⁺], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca²⁺] _i waves was also independent of influx of extracellular Ca²⁺. [Ca²⁺] _i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca²⁺] _i induced by different agonists were encoded into changes of baseline [Ca²⁺] _i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca²⁺] _i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca²⁺] _i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca²⁺] _i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar⁹,Met(O₂)¹¹]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.     

Acid secretion and the H,K ATPase of stomach.

The regulation of acid secretion was clarified by the development of H2-receptor antagonists in the 1970s. It appears that gastrin and acetylcholine exert their effects on acid secretion mainly by stimulation of histamine release from the enterochromaffin-like (ECL) cell of the fundic gastric mucosa. The isolated ECL cell of rat gastric mucosa responds to gastrin/cholecystokinin (CCK), acetylcholine, and epinephrine with histamine release and to somatostatin and R-alpha-methyl histamine by inhibition of histamine release. Histamine and acetylcholine stimulate the parietal cell by elevation of cAMP or [Ca]i by activation of H2 or M3 receptors, respectively. These independent pathways converge to activate the gastric acid pump, the H+,K+ ATPase. Activation is a function of the association of the ATPase with a potassium chloride transport pathway that occurs in the membrane of the secretory canaliculus of the parietal cell. Hence the secretory canaliculus is the site of acid secretion, the acid being pumped into the lumen of the canaliculus. The pump is composed of two subunits, a large catalytic and a smaller glycosylated protein. This final step of acid secretion has become the target of drugs also designed to inhibit acid secretion. The target domain of the benzimidazole class of acid pump inhibitors is the extracytoplasmic domain of the pump that is secreting acid, and the target amino acids are the cysteines present in this domain. The secondary structure of the pump can be analyzed by determining trypsin-sensitive bonds in intact, cytoplasmic-side-out vesicles of the ATPase, and it has been shown that the alpha subunit has at least eight membrane-spanning segments. Omeprazole, the first acid pump inhibitor, forms a disulfide bond with cysteines in the extracytoplasmic loop between the fifth and sixth membrane-spanning segment and to a cysteine in the extracytoplasmic loop between the seventh and eight segments, preventing phosphorylation of the pump by ATP. As a result of the effective and long-lasting inhibition of acid secretion by the acid pump inhibitor, superior clinical results have been found in all forms of acid-related disease.     

Autoinduction of differentiation in myeloid leukemic cells: restoration of normal coupling between growth and differentiation in leukemic cells that constitutively produce their own growth-inducing protein.

Growth and differentiation of normal myeloid haematopoietic cells are regulated by a family of macrophage- and granulocyte-inducing (MGI) proteins. Some of these proteins (MGI-1) induce cell growth and others (MGI-2) induce cell differentiation. Addition of MGI-1 to normal myeloid cells induces growth and also induces the endogenous production of MGI-2. This induction of differentiation-inducing protein by growth-inducing protein then ensures the coupling between growth and differentiation found in normal cells. There are myeloid leukemic cells that constitutively produce their own MGI-1, but the cells do not differentiate in culture medium containing horse or calf serum. By removing serum from the medium, or in medium with mouse or rat serum, these leukemic cells are induced to differentiate to mature cells, which like normal mature cells, then no longer multiply. Leukemic cells with constitutive production of MGI-1 continuously cultured in serum-free medium with transferrin were also induced to differentiate by removing transferrin. This induction of differentiation was in all these cases associated with the endogenous production of MGI-2 by the cells. The results indicate that changes in specific constituents of the culture medium can result in autoinduction of differentiation in these leukemic cells due to restoration of the induction of MGI-2 by MGI-1, which then restores the normal coupling of growth and differentiation.     

Photosynthesis as a function of short day induction and gibberellic Acid treatment in bougainvillea "san diego red"

Short day induction in Bougainvillea “San Diego Red” increases photosynthetic rates in mature leaves; gibberellic acid treatments, which inhibit flowering, cancel the short day effect. These results lend support of a nutritional hypothesis that suggests that in Bougainvillea assimilate supply to the reproductive axis increases before floral initiation and during flower development.     

Control mechanisms regulating gene expression during normal differentiation of myeloid leukemic cells: Differentiation defective mutants blocked in mRNA production and mRNA translation

Regulation of gene expression during myeloid cell differentiation has been analyzed using clones of myeloid leukemic cells that differ in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein MGI. Changes in the relative rate of synthesis for specific proteins were compared to changes in the relative amounts of corresponding translatable poly(A)⁺ mRNAs, assayed in the reticulocyte cell-free translation system, using two-dimensional gel electrophoresis. Of the 217 proteins which changed during MGI-induced differentiation of normally differentiating MGI⁺D⁺ leukemic cells, 136 could be identified as products of cell-free translation. Eighty-four percent of the 70 decreases in synthesis, most of which occurred early during differentiation, were not accompanied by a parallel decrease in the amount of translatable mRNA, but were accompanied by a parallel shift of the corresponding mRNAs from the polysomal to the monosomal and free mRNA fractions. These results indicate that most of the early decreases in the synthesis of proteins were translationally regulated. In contrast, 81% of the proteins which increased in synthesis and 71% of the proteins that were induced de novo were regulated at the level of mRNA production. Experiments with differentiation defective mutants have shown that they were blocked both at the level of mRNA production and mRNA translation. The data with these mutants have suggested that there were different subsets of translationally regulated proteins which were separately regulated. The translational blocks for several proteins in these mutant clones have also made it possible to identify additional translational sites of regulation for protein changes that were controlled at the level of mRNA production during normal differentiation. The results indicate that translational regulation may predominantly have a different function in cell differentiation than regulation by mRNA production, and that differentiation-defective mutants can be blocked at either level.