Synthesis of lens capsule and plasma membrane glycoproteins by lens epithelial cells and fibers in the rat

The lens of the eye possesses a capsule which is a greatly hypertrophied basement membrane. To investigate the synthesis of glycoproteins destined for this capsule, 3H-fucose was injected into the vitreous body of intact rats weighing approximately 200 gm. The animals were killed from 10 min to 14.5 months later, and their lenses were processed for electron microscope radioautography. At 10 min after injection, more than 58% of the silver grains were localized to the Golgi apparatus of the lens epithelial cells. By day 1, the heaviest sites of reaction were the plasma membrane (more than 50% of total label), the basal cytoplasm, and the adjacent lens capsule, where a heavy band of reaction was seen. The remainder of the capsule exhibited a lighter diffuse reaction. In the lens fibers, the label was at first localized to clusters of vesicles but then migrated to the plasma membrane and to the region of the capsule adjacent to the basal surface of these fibers. Light microscope radioautographs of the lens capsule at later time intervals revealed that by 1 month after injection the diffuse reaction had disappeared, and only the strongly labeled band remained. By 14.5 months after injection, this band had migrated partially across the lens capsule, but the capsule itself had increased considerably in thickness. On the other hand, the distance between the labeled band and the free edge of the capsule had decreased from that seen at the time of injection.     

Role of protein kinase C and the Na+/H+ antiporter in suppression of apoptosis by granulocyte macrophage colony-stimulating factor and interleukin-3.

Granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) suppress apoptosis in hemopoietic cells, a process of active cell death characterized by the degradation of genomic DNA into oligonucleosomic fragments. The present study was therefore initiated with the view that the two growth factors may trigger the same early events in the cell, leading to suppression of apoptosis. We provide evidence here for a role of protein kinase C and of the Na+/H+ antiporter in the signal transduction pathways activated by binding of GM-CSF or IL-3 to their respective receptors, resulting in suppression of apoptosis in target cells. First, kinetic studies indicate that the process is irreversible after two hours of deprivation. The suppression of apoptosis by GM-CSF and IL-3 is dose-dependent, with half-efficient concentrations that are in the range of the dissociation constants of the high affinity GM-CSF or IL-3 receptor, respectively. Second, the use of three inhibitors of protein kinase C (PKC), H7, staurosporine, and sphingosine, in concentrations that are below their toxicity limits, revert the suppression of apoptosis by IL-3 and GM-CSF. Conversely, the use of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, allows a bypass of receptor activation in suppression of apoptosis. Western blotting of cytosolic and membrane proteins indicate that exposure of the cells to GM-CSF, IL-3, or TPA results in translocation of PKC to the cell membrane. Our data, therefore, indicate that the activation of PKC is important in suppression of apoptosis by GM-CSF and IL-3. Third, the two amiloride derivatives 5-(N,N-hexamethylene) and 5-(N-ethyl-N-isopropyl)amiloride that specifically block the function of the Na+/H+ antiport also revert the protective effect of GM-CSF, IL-3, and TPA on MO7-E cells. Further, exposure of the cells to GM-CSF, IL-3, or TPA results in sustained pHi alkalinizatio, which is abrogated when the cells are preincubated with 5-(N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the antiport. Preincubation of the cells with staurosporine, a PKC inhibitor, also significantly reduces the effect of GM-CSF or IL-3 on pHi. Taken together, our data indicate that a functional antiport is required in suppression of apoptosis by GM-CSF, IL-3, or TPA. Furthermore, our results are consistent with the view that GM-CSF or IL-3 receptor activation initiates the sequential activation of PKC and of the Na+/H+ antiporter, resulting in suppression of apoptosis in target cells.     

Thermal refugia against coral bleaching throughout the northern Red Sea

Tropical reefs have been impacted by thermal anomalies caused by global warming that induced coral bleaching and mortality events globally. However, there have only been very few recordings of bleaching within the Red Sea despite covering a latitudinal range of 15° and consequently it has been considered a region that is less sensitive to thermal anomalies. We therefore examined historical patterns of sea surface temperature (SST) and associated anomalies (1982–2012) and compared warming trends with a unique compilation of corresponding coral bleaching records from throughout the region. These data indicated that the northern Red Sea has not experienced mass bleaching despite intensive Degree Heating Weeks (DHW) of >15°C‐weeks. Severe bleaching was restricted to the central and southern Red Sea where DHWs have been more frequent, but far less intense (DHWs <4°C‐weeks). A similar pattern was observed during the 2015–2016 El Niño event during which time corals in the northern Red Sea did not bleach despite high thermal stress (i.e. DHWs >8°C‐weeks), and bleaching was restricted to the central and southern Red Sea despite the lower thermal stress (DHWs < 8°C‐weeks). Heat stress assays carried out in the northern (Hurghada) and central (Thuwal) Red Sea on four key reef‐building species confirmed different regional thermal susceptibility, and that central Red Sea corals are more sensitive to thermal anomalies as compared to those from the north. Together, our data demonstrate that corals in the northern Red Sea have a much higher heat tolerance than their prevailing temperature regime would suggest. In contrast, corals from the central Red Sea are close to their thermal limits, which closely match the maximum annual water temperatures. The northern Red Sea harbours reef‐building corals that live well below their bleaching thresholds and thus we propose that the region represents a thermal refuge of global importance.     

Tiotropium bromide (Ba 679 BR), a novel long-acting muscarinic antagonist for the treatment of obstructive airways disease

Tiotropium bromide (Ba 679 BR) is a novel potent and long-lasting muscarinic antagonist that has been developed for the treatment of chronic obstructive airways disease (COPD). Binding studies with [3H]tiotropium bromide in human lung have confirmed that this is a potent muscarinic antagonist with equal affinity for M1-, M2- and M3-receptors and is approximately 10-fold more potent than ipratropium bromide. Tiotropium bromide dissociates very slowly from lung muscarinic receptors compared with ipratropium bromide. In vitro tiotropium bromide has a potent inhibitory effect against cholinergic nerve-induced contraction of guinea-pig and human airways, that has a slower onset than atropine or ipratropium bromide. After washout, however, tiotropium bromide dissociates extremely slowly compared with the dissociation of atropine and ipratropium bromide. Measurement of acetylcholine (ACh) release from guinea-pig trachea shows that tiotropium bromide, ipratropium bromide and atropine all increase ACh release on neural stimulation and that this effect is washed out equally quickly for the three antagonists. This confirms binding studies to transfected human muscarinic receptors which suggested that tiotropium bromide dissociates slowly from M3-receptors (on airway smooth muscle) but rapidly from M2 autoreceptors (on cholinergic nerve terminals). Clinical studies with inhaled tiotropium bromide confirm that it is a potent and long-lasting bronchodilator in COPD and asthma. Furthermore, it protects against cholinergic bronchoconstriction for > 24 h. This suggests that tiotropium bromide will be a useful bronchodilator, particularly in patients with COPD, and may be suitable for daily dosing. The selectivity for M3- over M2-receptors may also confer a clinical advantage.     

Surface display compared to periplasmic expression of a malarial antigen in Salmonella typhimurium and its implications for immunogenicity

Abstract Two different expression systems were investigated for the production of an 80 amino acid polypeptide, M3, from the C-terminus of the Plasmodium falciparum blood stage antigen Pf155/RESA in an attenuated Salmonella typhimurium vaccine strain. Upon expression, the malarial polypeptide was targeted either to the periplasm as a soluble fusion protein containing two IgG-binding domains (ZZ) from the staphylococcal protein A or, to the bacterial surface as an insert within a chimeric outer membrane protein A (OmpA) derived from Escherichia coli and Shigella dysenteriae . Both the ZZM3 and the OmpAM3 proteins were stably expressed in the periplasm or on the surface of Salmonella , respectively. The ZZ expression system yielded 10–100 times more malarial immunogen than did the OmpA system. Live recombinant Salmonella expressing ZZM3 or OmpAM3 were used to immunize mice intraperitoneally. Both the ZZM3 and OmpAM3 genes persisted for up to three weeks in bacteria isolated from different lymphoid organs. Bacteria expressing ZZM3 induced antibodies to M3, ZZ and to the Pf155/RESA antigen whereas, bacteria producing OmpAM3 induced similar levels of antibodies reactive with M3 but not with Pf155/RESA. Both recombinants induced a memory response of antibodies reactive with both M3 and Pf155/RESA. The high levels of M3 produced by the ZZ expression system make it suitable for the expression of heterologous antigens in Salmonella . Nevertheless, in spite of the quantitative difference in M3 expression, the ZZ and OmpA constructs elicited comparable immune responses to M3.     

Regulation of the calcium slow channel by cyclic GMP dependent protein kinase in chick heart cells

In order to assess the interaction between the cAMP-dependent and the cGMP-dependent phosphorylation pathways on the slow Ca²⁺ current (I_Ca(L)), whole-cell voltage-clamp experiments were conducted on embryonic chick heart cells. Addition of 8Br-cGMP to the bath solution reduced the basal (unstimulated) I_Ca(L). Intracellular application of the catalytic subunit of PK-A (PK-A(cat); 1.5 M) via the patch pipette rapidly potentiated I_Ca(L) by 215±16% (n=4); subsequent addition of 1 mM 8Br-cGMP to the bath reduced the amplitude of I_Ca(L) towards the initial control values (123±29%). Intracellular application of PK-G (25 nM pre-activated by 10^–7 M cGMP), rapidly inhibited the basal I_Ca(L) by 64±6% (n=8). Heat-denatured PK-G was ineffective. Subsequent additions of relatively high concentrations of 8Br-cAMP (1 mM) or isoproterenol (ISO, 1–10 M) did not significantly remove the PK-G blockade of I_Ca(L). The results of the present study suggest that: (a) 8Br-cGMP can inhibit the basal or stimulated (by PK-A(cat)) I_Ca(L) in embryonic chick myocardial cells. (b) PK-G applied intracellularly inhibits the basal I_Ca(L).     

The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy

Adams, G. R., and F. Haddad. The relationships amongIGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy. J. Appl. Physiol. 81(6):2509-2516, 1996.Insulin-like growth factor-1 (IGF-1) is known tohave anabolic effects on skeletal muscle cells. This study examined thetime course of muscle hypertrophy and associated IGF-1 peptide and mRNAexpression. Data were collected at 3, 7, 14, and 28 days after surgicalremoval of synergistic muscles of both normal and hypophysectomized(HX) animals. Overloading increased the plantaris (Plant) mass,myofiber size, and protein-to-body weight ratio in both groups (normaland HX; P < 0.05). Muscle IGF-1peptide levels peaked at 3 (normal) and 7 (HX) days of overloading withmaximum 4.1-fold (normal) and 6.2-fold (HX) increases. Increases inmuscle IGF-1 preceded the hypertrophic response. Total DNA content ofthe overloaded Plant increased in both groups. There was a strongpositive relationship between IGF-1 peptide and DNA content in theoverloaded Plant from both groups. These results indicate that1) the muscles from rats with bothnormal and severely depressed systemic levels of IGF-1 respond tofunctional overload with an increase in local IGF-1 expression and2) this elevated IGF-1 may becontributing to the hypertrophy response, possibly via the mobilizationof satellite cells to provide increases in muscle DNA.