In VivoP NMR Studies of Corn Root Tissue and Its Uptake of Toxic Metals

Excised corn root tissue has been evaluated for its viability, integrity of compartmentation, intracellular pH gradients, total mobile phosphorus content and nucleotide concentrations under different levels of acidity, and mineral stresses using in vivo³¹P nuclear magnetic resonance spectroscopy at 21 to 23°C. Perfusion with Al³⁺ ion at low pH (4.0) for 20 hours caused the overall concentration of nucleotides in the cytoplasm to decrease significantly relative to the control. Respiratory activity as measured by O₂ uptake decreased by a comparable amount over this time period. The addition of glucose to the Al-containing perfusate negated the inhibitory effects on the respiratory system. Treatment of the tissue with paramagnetic manganese ion while perfusing in the presence of O₂ allowed for the observation of the sequence of events leading to the irreversible trapping of Mn²⁺ in the vacuole. Pretreatment of the roots with Mg²⁺ prevented Mn²⁺ migration to the vacuole over the time period of this experiment. Hypoxia prevented all but a limited uptake of Mn²⁺ into the cytoplasm of the root tips. No evidence of Mn²⁺ complexation of either cytoplasmic or vacuole Pi suggests that the energy derived from O₂ consuming processes is necessary for the facilitated movement of this divalent cation.     

Effect of indomethacin on bombesin-like immunoreactivity, somatostatin and gastrin secretion from rat stomach

In the present study the effect of indomethacin-induced prostaglandin deficiency was examined on the release of bombesin-like immunoreactivity (BLI), a putative peptidergic neurotransmitter, from the isolated perfused rat stomach. In addition, gastrin and somatostatin (SLI) secretion was determined. Pretreatment of rats with indomethacin (2 mg/kg X h) resulted in a 3-fold increase of basal BLI secretion. In response to acetylcholine (2 X 10(-6) M) BLI rose from 2,000 to 4,000 pg/min, whereas in controls BLI increased from 400 to 1,400 pg/min. While absolute values for BLI secretion were higher in indomethacin-treated stomachs the relative increase above baseline was lower (100 vs. 250%). In control rats the increase in BLI secretion in response to acetylcholine was abolished when the acidity in the gastric lumen was increased from pH 7 to pH 2. After indomethacin, however, the stimulatory effect of acetylcholine during luminal pH 7 and pH 2 was identical. The decrease of SLI by acetylcholine at luminal pH 7 was abolished in indomethacin-treated stomachs in response to 10(-6) M acetylcholine, and 2 X 10(-6) M had even a stimulatory effect on SLI secretion. Indomethacin pretreatment reduced gastrin secretion at luminal pH 7. These data demonstrate that endogenous prostaglandins exert an inhibitory tone on basal and stimulated BLI and stimulated SLI secretion in the rat stomach. It is suggested that endogenous prostaglandins also inhibit the release of a peptidergic neurotransmitter, similar to their effect on the classical neurotransmitters acetylcholine and norepinephrine.     

Nucleosomal structure as probed by H3 histone thiol reactivity

Two H3 histone variants are found in equal amount in HeLa cells, and they have been characterized by two-dimensional gel electrophoresis followed by reaction with specific antibodies. These molecules are the only cysteine-containing histones, and they have been used as the target for thiol-specific reagents, in intact nuclei, isolated nucleosomes, histone complexes, and purified histones. Cysteine residues are available toN-ethylmaleimide only when histones are disassembled from the core particles. Upon reaction with these reagents, one of the H3 variants undergoes profound conformational changes, as revealed by an altered electrophoretic mobility.     

A budding and fusing journey through the secretory pathway. Genetic and In Vitro Analysis of Cell Compartmentalization: A UCLA symposium, Taos, NM, USA, February 3-9, 1990

Reconstitution of vesicular transport events and the molecular and genetic analysis of the secretory pathway have taken the field of membrane traffic into a new era. Already, proteins have been discovered that facilitate multiple transport steps, and studies of the identities and modes of action of additional transport components, such as those that specify the targets of transport vesicles, will soon follow. Even after we understand how transport vesicles form, find their targets, and then fuse, other fundamental questions will still remain. How are proteins sorted into distinct transport vesicles? How is the directionality of protein transport achieved? How do organelles maintain their identities in the face of large volumes of membrane traffic? Finally, how is membrane traffic regulated? Answers to each of these fundamental questions are likely to be available in the not-too-distant future.     

A novel protein related to cell cycle-dependent alterations of chromatin structure

We have detected a novel nuclear antigen, AF-2, which appears to be involved in cell cycle-dependent alterations of chromatin structure. Specific monoclonal antibodies detect the antigen spread over the whole cell during mitosis and in islet-like structures in the nuclei of a subpopulation of cells in interphase. Upon nucleolytic digestion of fixed cells, the antigen becomes available to the antibodies in all cells, indicating that AF-2 antigen is present during the whole cell cycle but differentially accessible. Digestion with the single strand specific S1 nuclease reveals that the alteration of chromatin structure induced by the introduction of nicks into the DNA rather than the digestion of DNA bound to the immunogenic epitope accounts for the change in accessibility of AF-2 antigen in interphase nuclei. The epitope recognized by the antibody in human cells is present in two polypeptides of 65 and 36 kDa, respectively, which are tightly bound to chromatin and cross-linkable to the nuclear matrix. The proteins also occur in the midbody during cytokinesis. The immunogenic epitope is conserved between man and fission yeast.     

Interferon-alpha down-regulates insulin receptors in lymphoblastoid (Daudi) cells. Relationship to inhibition of cell proliferation

The Daudi line of human lymphoblastoid cells requires insulin and transferrin for growth in serum-free medium and is highly sensitive to the inhibitory effect of human leukocyte interferon (IFN-alpha) on cell proliferation. A variant subline of Daudi cells, which is resistant to the antiproliferative action of IFN-alpha, also has been grown in serum-free medium containing insulin and transferrin. The proliferation of IFN-sensitive and -resistant Daudi cells is dependent on the occupancy of insulin receptors, with optimal cell proliferation observed at high receptor occupancy (nearly 100%). No evidence was found for receptors for insulin-like growth factor I on Daudi cells. IFN treatment of IFN-sensitive cells decreased the capacity of the cells to bind 125I-insulin. The altered binding capacity was due to diminished specific, lower affinity insulin binding, as detected at high 125I-insulin concentrations. Higher affinity insulin binding was not altered by IFN. Insulin binding was also reduced in detergent-solubilized extracts from IFN-treated sensitive Daudi cells and the magnitude of the effect was comparable to that observed in intact cells. This indicates that the total number of insulin binding sites (surface + internal) is decreased in IFN-treated sensitive cells. Insulin binding to IFN-sensitive cells decreased linearly with time between 6 and 48 h from the addition of IFN. The effect on lower affinity insulin binding developed more rapidly than the inhibitory effect of IFN on cell proliferation. The insulin-binding capacity of Daudi cells resistant to the antiproliferative effect of IFN was unaffected by IFN, despite the fact that these cells contain as many cell surface IFN receptors as sensitive cells. These observations raise the possibility that lower affinity insulin binding is important in the growth-promoting actions of insulin.